- Email: [email protected]
Protein kinase C and the regulation of the actin cytoskeleton
Cellular Signalling 18 (2006) 276 – 284 www.elsevier.com/locate/cellsig
Review
Protein kinase C and the regulation of the actin cytoskeleton Christer Larsson Lund University, Dept of Laboratory Medicine, Molecular Medicine, Entrance 78, 3rd floor, UMAS SE-205 02, Malmo¨ University Hospital, Malmo¨, Sweden Received 27 June 2005; received in revised form 18 July 2005; accepted 18 July 2005 Available online 16 August 2005
Abstract Protein kinase C (PKC) isoforms are central components in intracellular networks that regulate a vast number of cellular processes. It has long been known that in most cell types, one or more PKC isoforms influences the morphology of the F-actin cytoskeleton and thereby regulates processes that are affected by remodelling of the microfilaments. These include cellular migration and neurite outgrowth. This review focuses on the role of classical and novel PKC isoforms in migration and neurite outgrowth, and highlights some regulatory steps that may be of importance in the regulation by PKC of migration and neurite outgrowth. Many studies indicate that integrins are crucial mediators both upstream and downstream of PKC in inducing morphological changes. Furthermore, a number of PKC substrates, directly associated with the microfilaments, such as MARCKS, GAP43, adducin, fascin, ERM proteins and others have been identified. Their potential role in PKC effects on the cytoskeleton is discussed. D 2005 Elsevier Inc. All rights reserved. Keywords: Protein kinase C; Actin; Integrins; Cell movement; Neurite; Stress fibers
Contents 1. 2.
Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Protein kinase C and its effects on the actin cytoskeleton . . . . . . . . . . . . . . . 2.1. Phorbol esters influence cellular morphology . . . . . . . . . . . . . . . . . . 2.2. PKC isoforms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Protein kinase C isoforms involved in the regulation of cell spreading and migration . 4. Protein kinase C and neurite outgrowth . . . . . . . . . . . . . . . . . . . . . . . . 5. Mechanisms of PKC effects on cellular morphology — integrins and other receptors. 6. Mechanisms of PKC effects on cellular morphology — PKC substrates. . . . . . . . 6.1. MARCKS and GAP43 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2. Adducin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3. Fascin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.4. ERM proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.5. AFAP-110 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.6. Vinculin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.7. Summary of PKC substrates . . . . . . . . . . . . . . . . . . . . . . . . . . 7. PKC induction of stress fibres . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
E-mail address: [email protected]. 0898-6568/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.cellsig.2005.07.010
. . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . .
277 277 277 277 278 278 279 279 280 280 280 281 281 282 282 282 282 282 282
C. Larsson / Cellular Signalling 18 (2006) 276 – 284
1. Introduction Many morphological changes of a cell are at least partially driven by a remodelling of the actin cytoskeleton. There are many instances when it would be beneficial to be able to influence these changes. One example of detrimental effects of altered regulation of these processes is when a cell has acquired the capacity to migrate and to invade a tissue which is a prerequisite for a full-blown malignant phenotype of a cancer cell. In this case, it would be of benefit to be able to suppress the cytoskeletal activity. On the other hand, in some instances a possibility to facilitate the cytoskeletal processes would be advantageous. One such example concerns damaged axons in the central nervous system, which cannot regenerate spontaneously. A better understanding of the regulation of the cytoskeleton would thus facilitate our search for treatments for many diseases.
2. Protein kinase C and its effects on the actin cytoskeleton 2.1. Phorbol esters influence cellular morphology The morphology of the cytoskeleton is regulated by a large number of components and can be modified by many exogenous stimuli. It has for a long time been known that phorbol esters can stimulate or enhance cellular migration and motility [1 –6]. This is likely related to the more immediate effects on the actin cytoskeleton that also are observed upon addition of phorbol esters. These include cells spreading and ruffling [3,7] and dismantling of stress fibres [7– 9], although in some cell types stress fibres are formed upon phorbol ester exposure [10]. Phorbol esters are well-known activators of classical and novel protein kinase C (PKC) isoforms and it can therefore be assumed that one or several of these PKC isoforms promote changes in the cytoskeleton that facilitate or drive cell spreading and migration. However, phorbol esters bind and activate several other proteins that contain typical C1 domains [11] and are putative regulators of the microfilament morphology. These include the chimaerins which are GTPase-activating proteins and downregulate the activity of the Rac GTPase [12,13], the myotonic dystrophy kinaserelated Cdc42-binding kinase (MRCK) [14 – 16] and RasGRPs [17,18]. It is therefore likely that some phorbol ester-induced effects are mediated via one or several of these proteins. Nevertheless, the use of PKC inhibitors and other tools have clearly indicated that classical and novel PKC isoforms have significant roles in regulating cytoskeletondriven processes in cells. 2.2. PKC isoforms The PKC isoforms constitute a family of, depending on classification criteria, 10– 15 members [19]. In general,
277
classical (PKCa, hI, hII and g), novel (PKCy, q, D and u) and atypical (PKCN/E and ~) isoforms are considered as ‘‘proper’’ PKCs. Only classical and novel PKC isoforms contain C1 domains that bind phorbol esters and diacylglycerol (DAG). This review focuses on the role of classical and novel isoforms in the regulation of morphological changes driven by alterations in the F-actin structure. All PKC isoforms consist of one regulatory and one catalytic domain, with the regulatory domain containing binding sites for the PKC activators. The basic structure of the classical and novel PKCs is depicted in Fig. 1. Briefly, the regulatory domain of classical isoforms contains one Ca2+-binding C2 domain and two C1 domains which can bind DAG and phorbol esters. The general view of PKC activation has been that when the levels of Ca2+ and/or DAG increase in the cell they bind the C2 and C1 domains respectively, recruit the classical PKC to the plasma membrane and induce a conformational change of the enzyme. The kinase domain is thus exposed and can exert its catalytic function. A similar general mechanism is thought to operate for novel isoforms, with the exception that they are insensitive to Ca2+. Many studies have also shown that beside activation of PKC by membrane association, PKC is also regulated by interaction with other proteins, which will through interaction either modify the conformation of PKC or stabilise the activated conformation. The regulation of PKC has been reviewed in many papers, see for instance [20 – 22]. It is clear that the different isoforms have unique effects in cellular regulation. During the last 10 –15 years we have begun to understand how the different isoforms uniquely regulate specific cellular processes. This has been possible by using different approaches such as overexpression of isoforms, inhibitors with isoform specificity such as Go¨6976 and rottlerin, expression of dominant negative variants, cells with targeted deletion of one specific isoform or introduction of peptides that interfere with inhibitory intramolecular interactions or with interactions of PKC with other proteins. All these approaches have their advantages but also their limitations. The specificity of the inhibitors is clearly questionable, and the similarity between isoforms makes expression of large amounts of wild type PKCs, peptides, or Classical PKCs (α,βΙ,βΙΙ,γ) PS
C1a
C1b
C2
kinase
V5
Novel PKCs (δ,ε,η,θ) C2
PS
C1a
C1b
kinase
V5
Fig. 1. The overall structure of classical and novel PKC isoforms. The PKC isoforms consists of an N-terminal regulatory domain containing a pseudosubstrate (PS) two C1 domains (C1a and C1b) and one C2 domain and a C-terminal kinase domain with a V5 region that displays large variability between the isoforms. The C2 domain in classical isoforms binds Ca2+, whereas the same domain in novel isoforms does not bind Ca2+.
278
C. Larsson / Cellular Signalling 18 (2006) 276 – 284
dominant negative variants, not easy to interpret, due to the risk of unspecific effects. This can be at least partially circumvented by expressing analogous variants of several isoforms. Due to the limitations of the techniques some data on the importance of a certain isoform may therefore actually demonstrate what an isoform can do, rather than what an isoform really does.
3. Protein kinase C isoforms involved in the regulation of cell spreading and migration PKCa is perhaps the isoform that has emerged as a general promoter of cell spreading and migration. Elevating the levels of PKCa promotes migration of endothelial cells [23], MCF7 breast carcinoma cells [24] and 2C4 fibrosarcoma cells [25] and spreading of CHO cells [26] and PKCa levels correlate with the migratory capacity of colon carcinoma cells [27]. Furthermore, suppression of PKCa either by antisense approaches [28,29] or by dominant negative approaches [30] leads to impaired migration of vascular smooth muscle cells, endothelial cells and melanoma cells. However there are also indications that other isoforms promote migration. Downregulation or gene ablation of PKCh suppresses the migration of T cells on LFA-1 ligand [31] and MCP-1-stimulated chemotaxis of human monocytes [32]; and PKChII conceivably mediates the glucose potentiation of PDGF-stimulated migration of vascular smooth muscle cells [33] and PKChI contributes to cell migration on fibrinogen [34]. PKCy is important for stressinduced smooth muscle cell migration [35] and EGFstimulated motility of fibroblasts [36] and the levels of PKCy correlates to the metastatic potential of rat mammary carcinoma cell lines [37]. PKCq has been suggested to promote glioma cell migration [38] and it is necessary for hepatocyte growth factor-stimulated motility [39] and for cellular movement on fibronectin [40]. In addition, activated PKCq can rescue cell spreading in cells devoid of h1 integrin signalling [41]. There is also evidence for a role for PKCu. Kinase-inactive PKCu suppresses capillary endothelial cell migration [42]. Thus, a general effect of PKC in many cell types is to promote migration. In particular PKCa has this capacity but the relative contribution of different isoforms in vivo still needs to be firmly established.
4. Protein kinase C and neurite outgrowth The outgrowth of neurites, immature processes from neuronal cells that later may mature into axons or dendrites, is a crucial process for the differentiation of neurons. The outgrowth is led by the growth cone, a highly dynamic structure on the tip of the growing neurite. The morphology of the growth cone seems to be formed by similar changes in the F-actin structure as those that is seen in the spreading and migrating cell.
As for spreading and migration, phorbol esters have for a long time been known to have both positive and negative effects on neurite outgrowth. Initially it was seen that phorbol esters suppress neurite outgrowth from mouse neuroblastoma cells [43] and embryonic chick ganglia [44]. However, later studies have shown that the major effect may be to induce neurites exemplified by the effect on cultured human neuroblastoma cells [45], chick embryo sensory, sympathetic and parasympathetic ganglion cells [46,47], cerebellar granule cells [48] and on primary neurons cultured either on extracellular matrix proteins or on cadherins [49,50]. There are many studies indicating important roles for novel PKC isoforms in promoting neurite outgrowth. In PC12 cells, phorbol esters by themselves do not induce neurites but they potentiate neurite outgrowth induced by nerve growth factor (NGF), which is correlated to a translocation of PKCy [51] and PKCy has been suggested to mediated the activation of MAP kinases in PC12 upon neurogenic, but not mitogenic, stimulation [52]. On the other hand, increased levels of PKCq but not of PKCy potentiates neurite outgrowth induced by NGF or epidermal growth factor (EGF) from the same cells [53,54] and by itself induces neurites in cultured human neuroblastoma cells [55] and in immortalised neural precursor cells from the hippocampus and medullary raphe [56]. The latter effects are independent of the catalytic activity of PKC and mediated by the C1 domains. There is a motif N-terminal of the C1b domain that is conserved in all novel isoforms that is crucial for the neurite-inducing effect of PKC [57] suggesting that all novel isoforms may have the potential to induce neurites via this mechanism. Also classical isoforms may be of importance for some neurite-inducing stimuli such as the NCAM-mediated outgrowth of neurites [58,59]. The above mentioned studies describe more long term effects on the morphology of neuronal cells. When more local immediate effects on the growth cone are studied, PKC frequently turns out to mediate signals that either induce a collapse of the growth cone or have a repellent effect on its growth. Dopamine induces retraction of retinal horizontal cell neurites which seems to be mediated via PKC and can be mimicked by PKC activators [60] and the PTPA- and thrombin-induced growth cone collapse is conceivably transduced via PKCy [61] and PKCq [62] respectively and PKC inhibitors suppresses the repellent effects of myelin components on neurite outgrowth [63]. Furthermore, the CXCR4-induced repulsion of growth cones of both cerebellar granule cells and Xenopus spinal neurons are dependent on a classical PKC isoform [64]. Thus, a large number of studies indicate a general promoting effect in a long term perspective whereas PKC generally seems to, in the short term perspective, mediate growth cone collapse and retraction. These seemingly conflicting results may be due to the same protein having different short term and long term effects. Alternatively it
C. Larsson / Cellular Signalling 18 (2006) 276 – 284
may be related to the importance of different isoforms for the effects. The fact that PKC may mediate some effects on neurites via its regulatory domain further complicates the interpretation of the data.
5. Mechanisms of PKC effects on cellular morphology — integrins and other receptors The fact that there is no clear common effect of PKC isoforms for different cell types indicates that PKC may mediate its effects via several mechanisms. Such a conclusion is supported by the literature which indicates a wide range of pathways that may mediate the PKC effect, including direct phosphorylation of cytoskeletal regulators, influence on other signalling pathways and effects on receptors for extracellular matrix component. In this review the focus is on substrates directly involved in the modification of the actin cytoskeleton and on receptors of components of the extracellular matrix. Integrins constitute an important pool of the surface molecules that mediate the binding of the cell with the extracellular matrix. This interaction with the surrounding tissue not only acts as a structural element but also conveys signals into the cell. In order for a cell to move, there needs to be a constant replenishment of integrins to the front of the cell at the same time as integrins at the rear or at desmosomes need to be detached. There are now a large amount of studies that demonstrate an important role for PKC in the regulation of integrins and in the transduction of integrin signals. There are many reports of association and co-localisation of PKC isoforms with integrins. It is difficult to a discern a pattern of isoform-specific effects but many studies indicate that in particular PKCa [24,65,66] and/or PKCq [38,40] associates with h1 integrin. On the other hand, for h3 integrin, PKCh has been shown to be an important interacting partner [34]. For some of the interactions the binding may be indirect and dependent on RACK1 [34,38]. There is now considerable evidence that an important function of PKC is to regulate the transport and distribution of integrins. Increasing the levels of PKCa leads to more h1 integrins on the cellular surface and following PKC activation there is an internalisation of the integrins [24]. PKCa is also important for clustering-induced internalisation of a2/h1 integrins [67] and necessary for ligand induced internalisation of c-met, indicating that internalisation of receptors may be a common effect for PKCa. The internalisation of c-met is important for the activation of several transduction cascades and for cellular migration [39]. However, PKCa activity is not only important for the internalisation of integrins. PKCa-induced relocation of integrins often results in more integrins at the leading edge, which is in line with PKC having a pro-migratory effect. 12o-tetradecanoyl phorbol 13-acetate (TPA) stimulates the movement of PKC vesicles containing PKCa and h1
279
integrins to the leading edge, and disruption of the PKCa interaction with h1 integrins suppresses both TPA and EGFstimulated migration [65]. a6/h4 integrins are also targets for PKC. TPA induces transport of a6/h4 with tetraspanin 6.1 to the leading edge in pancreatic carcinoma [68] and it has been shown that PKCa phosphorylates h4 integrin [69] and leads to a relocation of a6/h4 from hemidesmosomes to the leading edge [70] during both TPA- and EGF-stimulated migration of A431 cells. The PKC-mediated translocation of integrins can be mediated by other receptors for extracellular matrix components. One cell surface receptor that is of particular interest in this aspect is syndecan-4. This is a proteoglycan that binds several extracellular matrix proteins and upon ligation in the presence of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] activates PKCa. Binding to syndecan-4 is actually sufficient to induce a h1 integrin-dependent cell spreading and stress fibre formation. This is mediated via PKCa [26]. The adhesion of cells to integrin ligands frequently makes the cell spread and in many instances it also makes the cell migrate. It is conceivable that PKC in a lot of these cases is crucial for the transduction of the signal from integrin ligation to cell spreading. T-cells devoid of PKCh do not migrate on LFA-1 ligand and migration can be rescued by expression of PKChI [31] and spreading of PKCh null platelets on fibrinogen is suppressed compared to wild type platelets [34]. Furthermore, PKCq null fibroblasts have impaired migration on fibronectin [40]. PKC is not only crucial for integrin-mediated effects in some cell types. Activation of PKC can actually rescue some characteristics that are lost in cells in which integrin signalling has been abrogated. PKCq rescues cell spreading on fibrinogen and collagen I of cells in which signalling through h1 integrins had been destroyed [41] and activation of either PKCa, PKCy or PKCq rescues spreading of a5 integrin-deficient cells on fibronectin [71]. In some cases, PKC complements integrin ligation and potentiates the morphological effects that is dependent on or mediated via integrins. For melanoma cells, binding to fibronectin via a5h1 integrins leads to spreading and further stimulation of syndecan-4 activates PKCa with the formation of focal adhesions subsequent cellular migration as result. However, migration and formation of focal adhesions, stimulated by spreading on a4h1 integrin ligands are independent of PKC [30]. An effect along the same line is seen in CHO cells expressing a2bh3 integrins. These cells weakly adhere to antibodies, but addition of TPA makes the cell spread [4].
6. Mechanisms of PKC effects on cellular morphology — PKC substrates It seems clear that modification of integrin function, control of integrin localisation and transduction of integrin
280
C. Larsson / Cellular Signalling 18 (2006) 276 – 284
signals seems to be important mechanisms for PKC effects on the cytoskeleton. Other components that are conceivably central in the mediation of the PKC effects are the substrates of PKC that have been shown to directly influence the morphology of the actin cytoskeleton. There are a number of such substrates described; some were identified more than twenty years ago whereas some were recently characterised. Here some of them are discussed. 6.1. MARCKS and GAP43 This family of related proteins consisting of MARCKS, MacMARCKS, GAP43 and CAP23 have long been candidates for mediating PKC effects on the cytoskeleton. MARCKS was one of the first PKC substrates that was characterised. PKC-mediated phosphorylation of an 87 kD protein was described already in 1982 [72] and the protein was later identified as MARCKS [73]. MacMARCKS, a closely related protein, was isolated shortly thereafter [74,75]. MARCKS is a membrane-bound protein. The membrane interaction is dependent on both an N-terminal myristoylation of MARCKS and on a cluster of basic residues in its effector domain. The myristic acid is inserted into membranes and the basic residues interact with negatively charged head groups of membrane phospholipids. The PKC phosphorylation sites are located in the effector domain and phosphorylation alters the net charge of the domain, which results in a dissociation of MARCKS from the membrane [76]. MARCKS phosphorylation can be carried out by both classical and novel PKC isoforms [77]. GAP43 is an analogous protein with an effector domain and N-terminal palmitoylation. It was initially detected as a phosphoprotein in synaptosomes [78] and independently as a substrate of PKC, the phosphorylation of which correlated to synaptic plasticity [79]. Depletion of GAP43 markedly alters neurite and growth cone morphology [80] and overexpression of GAP43 in the nervous system promotes neurite sprouting, an effect that is dependent on its PKC phosphorylation sites [81]. As for MARCKS, GAP43 can be phosphorylated by classical and novel isoforms [82]. MARCKS has been suggested to be an important factor for the anchoring of the actin cytoskeleton to the plasma membrane [83]. A dissociation of MARCKS from the membrane would thereby lead to a detachment of microfilaments from the membrane. However, there are later indications that MARCKS exerts its functions by sequestering PI(4,5)P2 [84,85] and a release of MARCKS from the membrane would then lead to an increase in available PI(4,5)P2. This mechanism appears to be common for MARCKS, CAP23 and GAP43 and primarily takes place at rafts rich in PI(4,5)P2 [86]. GAP43, in a manner dependent on its phosphorylation status has also been shown to affect the structure of actin filaments with the phosphorylated variant promoting the stabilisation of long filaments [87]. There are clear indications that phosphorylation of MARCKS proteins are important for some PKC effects on
the cytoskeleton. Dominant negative, i.e. non-phosphorylatable variants, MacMARCKS [88] and MARCKS [89] suppress phorbol ester-mediated cell spreading. It seems like a dynamic relocation of MARCKS is necessary for the spreading and subsequent formation of focal adhesions in myoblasts seeded on fibronectin. During spreading there is a PKC-dependent phosphorylation and translocation to the cytosol, whereas later MARCKS translocates to the focal adhesions that are under assembly [71,90].This may thus reflect a role for MARCKS in regulating the local F-actin structure. PKC phosphorylation of MacMARCKS has been shown to be important for TPA-mediated lateral diffusion of h2 integrins [91], suggesting that MacMARCKS may also anchor integrins. A plausible effect of PKC phosphorylation of MARCKS and GAP43 is thus an increased availability of PI(4,5)P2 in the plasma membrane which can result in a dynamic reorganisation of the cortical microfilaments. However, the PKC effect is conceivably not limited to an ‘‘inactivation’’ of the sequestering of PI(4,5)P2. There are, as mentioned above, experimental evidence demonstrating that PKC phosphorylation must give also other functions to these proteins, than merely ablate their binding to PI(4,5)P2. 6.2. Adducin Adducin is a protein that simultaneously caps F-actin and assembles F-actin with spectrin, a function that is fundamental for the shape of the erythrocyte [92]. PKC phosphorylates adducin from erythrocytes [93], brain [94] and fibroblasts [95], which leads to a diminished activity of adducin in terms of both F-actin-capping and promotion of spectrin-binding to F-actin [96]. A PKC-mediated loss of adducin interactions with F-actin and spectrin, resulting in exposure of free barbed ends, is conceivably important for the PKC-mediated morphological changes that take place during platelet activation [97]. Phosphorylated adducin is also found in dendritic spines, which are dynamic structures, indicating a role in cytoskeletal remodelling [96]. 6.3. Fascin Fascin is a protein that tightly bundles F-actin and is important for the formation of actin-based protrusions and for maintaining cytoplasmic F-actin bundles. The bundling activity of fascin is dependent on its capacity to crosslink actin monomers and this is inhibited by phosphorylation of S39 [98]. The phosphorylation status of this site is dependent on the extracellular matrix. When C2C12 myoblasts are cultured on thrombospondin-1 the site is dephosphorylated but seeding onto fibronectin induces a PKC-mediated phosphorylation of S39 [99]. The phosphorylation is conceivably mediated by PKCa [100] and it leads to the dissociation of fascin from the F-actin bundles and a diffuse localisation of fascin. This is likely of importance for cell spreading since a S39A fascin mutant
C. Larsson / Cellular Signalling 18 (2006) 276 – 284
suppresses spreading on fibronectin whereas a S39D mutant promotes it [99]. However, the role of the PKCa-fascin connection seems to be more elusive. PKCa can directly bind phosphorylated fascin and disruption of this interaction suppresses MCF-7 cell spreading but actually potentiates the cell migration on fibronectin and the formation of focal adhesions [100]. Thus, when seeded on fibronectin PKCa phosphorylates fascin which dissociates from F-actin and thereby releases the tight bundles. This is important for optimal spreading. PKCa binds the phosphorylated fascin and the complex is important for fine-tuning of the migratory response. It will be of interest to know if fascin targets PKC to other substrates or whether the main function of the persistent interaction is to maintain fascin in a phosphorylated state. 6.4. ERM proteins The ERM (ezrin-radixin-moesin) proteins constitute an interesting group of proteins that both function as connectors of the microfilaments with the plasma membrane as well as transduce signals to different transduction pathways. ERM proteins are maintained in a dormant state by an intramolecular interaction between the N-terminal FERM domain and the C-terminus. This interaction, with a subsequent conformational release of the protein, can be disrupted by phosphorylation of a threonine residue in the C-terminus [101]. For moesin, PKCu has been shown to phosphorylate this threonine (T558) residue [102] with a consequent exposure of both the N- and C-termini and increased interaction with cortical microfilaments [103]. In addition, PKCa has been shown to bind to and phosphorylate the corresponding residue in ezrin. The proteins colocalise in the leading edge of migrating cells and the phosphorylation is likely of functional importance since overexpression of T558A ezrin mutant suppresses migration in PKCa-overexpressing cells [25]. The activation of selected ERM proteins may therefore be an important pathway in mediating PKC effects on migration. The ERM proteins also interact with adhesion molecules including CD44 and ICAMs [101]. This may be another site for PKC action. PKC activation leads to a dephosphorylation of S325 at same time as it phosphorylates S291 in CD44. This alteration of phosphorylation decreases the interaction of CD44 with ezrin and a S291A mutant suppresses TPA-induced migration [104]. Thus, PKC can influence ERM protein activities both by altering their conformation and by determining the binding capacity of their interaction partners. 6.5. AFAP-110 AFAP-110 is a multi-domain protein that may convey some PKC signals to the cytoskeleton. AFAP-110 can both cross-link F-actin and bind Src which results in the activation of Src. Classical PKCs bind to AFAP-110 and
281
phosphorylates it and disrupts its multimerisation [105] and makes it able to activate Src [106]. Expression of an AFAP110 mutant with deleted PKC-binding site suppresses TPAinduced ruffling indicating a role for AFAP-110 in the TPA effect [105]. AFAP-110 also seems to be important for the formation of podosomes [106]. It remains to be seen if all AFAP-110-mediated effects are due to the subsequent MARCKS/GAP43 PKC
Adducin PKC
Fascin PKC
ERM proteins PKC
Fig. 2. A rough sketch of the effects of PKC phosphorylation on the function of some PKC substrates. PKC substrates are depicted in green, Factin in red, PI(4,5)P2 in blue, other proteins in orange and yellow and the plasma membrane is black. MARCKS/GAP43: The unphosphorylated protein sequesters PI(4,5)P2 and may also couple F-actin to the plasma membrane. These functions are lost upon phosphorylation. The free PI(4,5)P2 will recruit actin-modifying proteins and the net effect will be to increase actin assembly [115]. Adducin: Adducin caps the barbed end of F-actin and supports the interaction of F-actin with spectrin. A phosphorylation by PKC results in a dissociation of the complex and the exposure of free barbed ends on which actin polymerisation can take place. Fascin: Fascin bundles F-actin in tight structures. The binding to actin is lost upon PKC phosphorylation of fascin leading to the dissociation of the bundles. ERM proteins: In the resting state, these proteins are localised in the cytosol and kept in a closed conformation by an intramolecular interaction between the N-terminal FERM domain and the C-terminal part of the protein. PKC phosphorylation disrupts this interaction and opens up the conformation. The exposed FERM domain interacts with plasma membrane proteins and the C-terminus can bind F-actin. In the open conformation, ERM proteins can interact with many signalling proteins and thereby focus further signalling to the plasma membrane.
282
C. Larsson / Cellular Signalling 18 (2006) 276 – 284
activation of Src or if any of them are related to direct effects on the F-actin structure.
to the RhoA pathway. PKCa has been shown to mediate thrombin-induced activation of p115RhoGEF, an activator of RhoA, and induction of stress fibres [114].
6.6. Vinculin Vinculin is a PKC substrate [107] and PKCa has been shown to bind to and phosphorylate vinculin [108]. This may explain why in some cell systems the translocation of vinculin to focal adhesions and the formation of different cellular adhesions are dependent on PKC [109], in particular on PKCa [110]. 6.7. Summary of PKC substrates Several of the above listed substrates may contribute to PKC-induced spreading and migration. These events require a modification of the cortical cytoskeleton and phosphorylation of MARCKS and/or GAP43 will cause the release of PI(4,5)P2 and thereby a local accumulation of F-actin modifying proteins. Phosphorylation of adducin will lead to a dissociation of spectrin from F-actin and exposure of free barbed ends. PKC activation will likewise make fascin loose its bundling activity leading to a less rigid F-actin structure and ERM proteins may be recruited to plasma membrane proteins and interact with F-actin or change the activity of other signalling proteins. Thus, there are several mechanisms through which a local activation of PKC may lead to dynamic changes in the local cortical cytoskeleton. These events are summarised in Fig. 2.
8. Conclusions It is clear that PKC regulates the actin cytoskeleton in a wide range of cell types and a general activation of PKC isoforms leads to suppression of stress fibres, formation of ruffles and a pro-migratory effect in most cells that have been studied. However, PKC activation can also lead to formation of stress fibres in some cell types. This may be related to specific and different effects of the PKC isoforms. A general impression of the literature is that particularly PKCa has the capacity to alter the cytoskeleton in a promigratory manner and to promote migration and that PKCy is involved in the RhoA pathway. This may be due to the fact that primarily these isoforms have been studied and that other PKCs could have the same effects in the experimental systems. It will be of interest to see which isoforms that mediate the effects in vivo. A large number of PKC substrates have been identified and are conceivable candidates for mediating the PKC effects on the cytoskeleton. However, there are undoubtedly substrates that remain to be characterised and also interacting proteins that are of importance for the PKC effects.
Acknowledgements 7. PKC induction of stress fibres As discussed above, in most cell types a general PKC activation leads to a loss of stress fibres and induction of ruffles and an increased tendency to migration. However, there are also cases when PKC seems to promote or drive the formation of stress fibres. The formation of focal adhesions and stress fibres in fibroblasts that have spread on fibronectin is dependent on and can be induced by PKC [111]. The leukotrine-induced stress fibre formation in intestinal epithelial cells is for instance mediated by PKC, presumably PKCy, and the effect can be obtained by a direct activation of PKC [10]. Leukotriene-stimulated PKC activation seems to be mediated by RhoA and there are cases when PKCy is downstream of RhoA or mimicks RhoA activation. Rho/ ROCK-mediated MARCKS phosphorylation in endocrine cells is dependent on PKCy [112] and PKCy activation is crucial for EGF-induced phosphorylation of the myosin light chain in fibroblasts [36]. The facts that overexpression of PKCy in endothelial cells make them more prone to form focal adhesion and suppresses their migratory tendency, the opposite to what is observed with PKCa [23], and that RhoA induces focal adhesions that contain PPKCy [113] also supports a role for PKCy in mediating or inducing RhoA effects. However, it is not only PKCy that has been coupled
This work is supported by the Swedish Cancer Society, the Swedish Research Council, the Swedish Children’s Cancer Foundation, Malmo¨ University Hospital Research funds and the Crafoord, Greta and Johan Kock, and Ollie and Elof Ericsson Foundations.
References [1] [2] [3] [4]
[5] [6] [7] [8] [9] [10] [11] [12]
J. Varani, S.E. Fligiel, P. Perone, Int. J. Cancer 35 (1985) 559. R. Sears, G. Ciment, Dev. Biol. 130 (1988) 133. K. Vuori, E. Ruoslahti, J. Biol. Chem. 268 (1993) 21459. P. Defilippi, M. Venturino, D. Gulino, A. Duperray, P. Boquet, C. Fiorentini, G. Volpe, M. Palmieri, L. Silengo, G. Tarone, J. Biol. Chem. 272 (1997) 21726. F. Entschladen, B. Niggemann, K. Zanker, P. Friedl, J. Immunol. 159 (1997) 3203. V. Rigot, M. Lehmann, F. Andre, N. Daemi, J. Marvaldi, J. Luis, J. Cell Sci. 111 (1998) 3119. M. Schliwa, T. Nakamura, K.R. Porter, U. Euteneuer, J. Cell Biol. 99 (1984) 1045. A.J. Ridley, A. Hall, EMBO J. 13 (1994) 2600. D. Brandt, M. Gimona, M. Hillmann, H. Haller, H. Mischak, J. Biol. Chem. 277 (2002) 20903. R. Massoumi, C. Larsson, A. Sjo¨lander, J. Cell Sci. 115 (2002) 3509. D. Ron, M.G. Kazanietz, FASEB J. 13 (1999) 1658. S. Ahmed, R. Kozma, C. Monfries, C. Hall, H.H. Lim, P. Smith, L. Lim, Biochem. J. 272 (1990) 767.
C. Larsson / Cellular Signalling 18 (2006) 276 – 284 [13] R. Kozma, S. Ahmed, A. Best, L. Lim, Mol. Cell Biol. 16 (1996) 5069. [14] T. Leung, X.Q. Chen, I. Tan, E. Manser, L. Lim, Mol. Cell Biol. 18 (1998) 130. [15] I. Tan, K.T. Seow, L. Lim, T. Leung, Mol. Cell. Biol. 21 (2001) 2767. [16] S. Wilkinson, H.F. Paterson, C.J. Marshall, Nat. Cell Biol. 7 (2005) 255. [17] J.O. Ebinu, D.A. Bottorff, E.Y. Chan, S.L. Stang, R.J. Dunn, J.C. Stone, Science 280 (1998) 1082. [18] C.E. Tognon, H.E. Kirk, L.A. Passmore, I.P. Whitehead, C.J. Der, R.J. Kay, Mol. Cell Biol. 18 (1998) 6995. [19] H. Mellor, P.J. Parker, Biochem. J. 332 (1998) 281. [20] A.C. Newton, Chem. Rev. 101 (2001) 2353. [21] D. Schechtman, D. Mochly-Rosen, Oncogene 20 (2001) 6339. [22] Y. Shirai, N. Saito, J. Biochem. (Tokyo) 132 (2002) 663. [23] E.O. Harrington, J. Loffler, P.R. Nelson, K.C. Kent, M. Simons, J.A. Ware, J. Biol. Chem. 272 (1997) 7390. [24] T. Ng, D. Shima, A. Squire, P.I.H. Bastiaens, S. Gschmeissner, M.J. Humphries, P.J. Parker, EMBO J. 18 (1999) 3909. [25] T. Ng, M. Parsons, W.E. Hughes, J. Monypenny, D. Zicha, A. Gautreau, M. Arpin, S. Gschmeissner, P.J. Verveer, P.I.H. Bastiaens, P.J. Parker, EMBO J. 20 (2001) 2723. [26] C.K. Thodeti, R. Albrechtsen, M. Grauslund, M. Asmar, C. Larsson, Y. Takada, A.M. Mercurio, J.R. Couchman, U.M. Wewer, J. Biol. Chem. 278 (2003) 9576. [27] K. Masur, K. Lang, B. Niggemann, K.S. Zanker, F. Entschladen, Mol. Biol. Cell 12 (2001) 1973. [28] H. Haller, C. Lindschau, C. Maasch, H. Olthoff, D. Kurscheid, F.C. Luft, Circ. Res. 82 (1998) 157. [29] A. Wang, M. Nomura, S. Patan, J.A. Ware, Circ. Res. 90 (2002) 609. [30] Z. Mostafavi-Pour, J.A. Askari, S.J. Parkinson, P.J. Parker, T.T.C. Ng, M.J. Humphries, J. Cell Biol. 161 (2003) 155. [31] Y. Volkov, A. Long, S. McGrath, D. Ni Eidhin, D. Kelleher, Nat. Immunol. 2 (2001) 508. [32] K.A. Carnevale, M.K. Cathcart, J. Biol. Chem. 278 (2003) 25317. [33] M. Campbell, E.R. Trimble, Circ. Res. 96 (2005) 197. [34] C.S. Buensuceso, A. Obergfell, A. Soriani, K. Eto, W.B. Kiosses, E.G. Arias-Salgado, T. Kawakami, S.J. Shattil, J. Biol. Chem. 280 (2005) 644. [35] C. Li, F. Wernig, M. Leitges, Y. Hu, Q. Xu, FASEB J. 17 (2003) 2106. [36] A. Iwabu, K. Smith, F.D. Allen, D.A. Lauffenburger, A. Wells, J. Biol. Chem. 279 (2004) 14551. [37] S.C. Kiley, K.J. Clark, S.K. Duddy, D.R. Welch, S. Jaken, Oncogene 18 (1999) 6748. [38] A. Besson, T.L. Wilson, V.W. Yong, J. Biol. Chem. 277 (2002) 22073. [39] S. Kermorgant, D. Zicha, P.J. Parker, EMBO J. 23 (2004) 3721. [40] J. Ivaska, R.D.H. Whelan, R. Watson, P.J. Parker, EMBO J. 21 (2002) 3608. [41] A.L. Berrier, A.M. Mastrangelo, J. Downward, M. Ginsberg, S.E. LaFlamme, J. Cell Biol. 151 (2000) 1549. [42] S. Tang, K.G. Morgan, C. Parker, J.A. Ware, J. Biol. Chem. 272 (1997) 28704. [43] D.N. Ishii, Cancer Res. 38 (1978) 3886. [44] D.N. Ishii, E. Fibach, H. Yamasaki, I.B. Weinstein, Science 200 (1978) 556. [45] S. Pa˚hlman, L. Odelstad, E. Larsson, G. Grotte, K. Nilsson, Int. J. Cancer 28 (1981) 583. [46] L. Hsu, D. Natyzak, J.D. Laskin, Cancer Res. 44 (1984) 4607. [47] L. Hsu, Neurosci. Lett. 62 (1985) 283. [48] M.A. Cambray-Deakin, J. Adu, R.D. Burgoyne, Brain Res. Dev. Brain Res. 53 (1990) 40. [49] J.L. Bixby, P. Jhabvala, J. Cell Biol. 111 (1990) 2725. [50] J.L. Bixby, Neuron 3 (1989) 287. [51] K.R. O’Driscoll, K.K. Teng, D. Fabbro, L.A. Greene, I.B. Weinstein, Mol. Cell. Biol. 6 (1995) 449.
283
[52] K.C. Corbit, D.A. Foster, M.R. Rosner, Mol. Cell. Biol. 19 (1999) 4209. [53] B. Hundle, T. McMahon, J. Dadgar, R.O. Messing, J. Biol. Chem. 270 (1995) 30134. [54] C. Brodie, K. Bogi, P. Acs, P. Lazarovici, G. Petrovics, W.B. Anderson, P.M. Blumberg, Cell Growth Differ. 10 (1999) 183. [55] R. Zeidman, B. Lo¨fgren, S. Pa˚hlman, C. Larsson, J. Cell Biol. 145 (1999) 713. [56] M. Ling, U. Trolle´r, R. Zeidman, C. Lundberg, C. Larsson, Exp. Cell Res. 292 (2004) 135. [57] M. Ling, U. Troller, R. Zeidman, H. Stensman, A. Schultz, C. Larsson, J. Biol. Chem. 280 (2005) 17910. [58] I. Leshchyns’ka, V. Sytnyk, J.S. Morrow, M. Schachner, J. Cell Biol. 161 (2003) 625. [59] K. Kolkova, H. Stensman, V. Berezin, E. Bock, C. Larsson, J. Neurochem. 92 (2005) 886. [60] S. Rodrigues Pdos, J.E. Dowling, Proc. Natl. Acad. Sci. U. S. A. 87 (1990) 9693. [61] S.E. Ensslen, S.M. Brady-Kalnay, Mol. Cell. Neurosci. 25 (2004) 558. [62] K. Mikule, S. Sunpaweravong, J.C. Gatlin, K.H. Pfenninger, J. Biol. Chem. 278 (2003) 21168. [63] R. Sivasankaran, J. Pei, K.C. Wang, Y.P. Zhang, C.B. Shields, X.M. Xu, Z. He, Nat. Neurosci. 7 (2004) 261. [64] Y. Xiang, Y. Li, Z. Zhang, K. Cui, S. Wang, X.B. Yuan, C.P. Wu, M.M. Poo, S. Duan, Nat. Neurosci. 5 (2002) 843. [65] M. Parsons, M.D. Keppler, A. Kline, A. Messent, M.J. Humphries, R. Gilchrist, I.R. Hart, C. Quittau-Prevostel, W.E. Hughes, P.J. Parker, T. Ng, Mol. Cell. Biol. 22 (2002) 5897. [66] K. Podar, Y.-T. Tai, B.K. Lin, R.P. Narsimhan, M. Sattler, T. Kijima, R. Salgia, D. Gupta, D. Chauhan, K.C. Anderson, J. Biol. Chem. 277 (2002) 7875. [67] P. Upla, V. Marjomaki, P. Kankaanpaa, J. Ivaska, T. Hyypia, F.G. van der Goot, J. Heino, Mol. Biol. Cell 15 (2004) 625. [68] M. Herlevsen, D.-S. Schmidt, K. Miyazaki, M.J. Zoller, Cell Sci. 116 (2003) 4373. [69] I. Rabinovitz, L. Tsomo, A.M. Mercurio, Mol. Cell. Biol. 24 (2004) 4351. [70] I. Rabinovitz, A. Toker, A.M. Mercurio, J. Cell Biol. 146 (1999) 1147. [71] M.-H. Disatnik, S.C. Boutet, C.H. Lee, D. Mochly-Rosen, T.A. Rando, J. Cell Sci. 115 (2002) 2151. [72] W.C. Wu, S.I. Walaas, A.C. Nairn, P. Greengard, Proc. Natl. Acad. Sci. U. S. A. 79 (1982) 5249. [73] D.J. Stumpo, J.M. Graff, K.A. Albert, P. Greengard, P.J. Blackshear, Proc. Natl. Acad. Sci. U. S. A. 86 (1989) 4012. [74] P.J. Blackshear, G.M. Verghese, J.D. Johnson, D.M. Haupt, D.J. Stumpo, J. Biol. Chem. 267 (1992) 13540. [75] J. Li, A. Aderem, Cell 70 (1992) 791. [76] M. Thelen, A. Rosen, A.C. Nairn, A. Aderem, Nature 351 (1991) 320. [77] A. Fujise, K. Mizuno, Y. Ueda, S. Osada, S. Hirai, A. Takayanagi, N. Shimizu, M.K. Owada, H. Nakajima, S. Ohno, J. Biol. Chem. 269 (1994) 31642. [78] A.B. Oestreicher, H. Zwiers, P. Schotman, W.H. Gispen, Brain Res. Bull. 6 (1981) 145. [79] R.F. Akers, A. Routtenberg, Brain Res. 334 (1985) 147. [80] L. Aigner, P. Caroni, J. Cell Biol. 123 (1993) 417. [81] L. Aigner, S. Arber, J.P. Kapfhammer, T. Laux, C. Schneider, F. Botteri, H.R. Brenner, P. Caroni, Cell 83 (1995) 269. [82] S.A. Oehrlein, P.J. Parker, T. Herget, Biochem. J. 317 (1996) 219. [83] J.H. Hartwig, M. Thelen, A. Rosen, P.A. Janmey, A.C. Nairn, A. Aderem, Nature 356 (1992) 618. [84] M. Glaser, S. Wanaski, C.A. Buser, V. Boguslavsky, W. Rashidzada, A. Morris, M. Rebecchi, S.F. Scarlata, L.W. Runnels, G.D. Prestwich, J. Chen, A. Aderem, J. Ahn, S. McLaughlin, J. Biol. Chem. 271 (1996) 26187.
284
C. Larsson / Cellular Signalling 18 (2006) 276 – 284
[85] J. Wang, A. Arbuzova, G. Hangyas-Mihalyne, S. McLaughlin, J. Biol. Chem. 276 (2001) 5012. [86] T. Laux, K. Fukami, M. Thelen, T. Golub, D. Frey, P. Caroni, J. Cell Biol. 149 (2000) 1455. [87] Q. He, E.W. Dent, K.F. Meiri, J. Neurosci. 17 (1997) 3515. [88] J. Li, Z. Zhu, Z. Bao, J. Biol. Chem. 271 (1996) 12985. [89] M.M. Myat, S. Anderson, L.A. Allen, A. Aderem, Curr. Biol. 7 (1997) 611. [90] M.-H. Disatnik, S.C. Boutet, W. Pacio, A.Y. Chan, L.B. Ross, C.H. Lee, T.A. Rando, J. Cell Sci. 117 (2004) 4469. [91] X. Zhou, J. Li, J. Biol. Chem. 275 (2000) 20217. [92] Y. Matsuoka, X. Li, V. Bennett, Cell. Mol. Life Sci. 57 (2000) 884. [93] E. Ling, K. Gardner, V. Bennett, J. Biol. Chem. 261 (1986) 13875. [94] V. Bennett, K. Gardner, J.P. Steiner, J. Biol. Chem. 263 (1988) 5860. [95] A. Waseem, H.C. Palfrey, J. Cell Sci. 96 (1990) 93. [96] Y. Matsuoka, X. Li, V. Bennett, J. Cell Biol. 142 (1998) 485. [97] K.L. Barkalow, J.E. Italiano Jr., D.E. Chou, Y. Matsuoka, V. Bennett, J.H. Hartwig, J. Cell Biol. 161 (2003) 557. [98] J.C. Adams, Curr. Opin. Cell Biol. 16 (2004) 590. [99] J.C. Adams, J.D. Clelland, G.D.M. Collett, F. Matsumura, S. Yamashiro, L. Zhang, Mol. Biol. Cell 10 (1999) 4177. [100] N. Anilkumar, M. Parsons, R. Monk, T. Ng, J.C. Adams, EMBO J. 22 (2003) 5390. [101] A. Bretscher, K. Edwards, R.G. Fehon, Nat. Rev. Mol. Cell Biol. 3 (2002) 586.
[102] S.F. Pietromonaco, P.C. Simons, A. Altman, L. Elias, J. Biol. Chem. 273 (1998) 7594. [103] P.C. Simons, S.F. Pietromonaco, D. Reczek, A. Bretscher, L. Elias, Biochem. Biophys. Res. Commun. 253 (1998) 561. [104] J.W. Legg, C.A. Lewis, M. Parsons, T. Ng, C.M. Isacke, Nat. Cell Biol. 4 (2002) 399. [105] Y. Qian, J.M. Baisden, L. Cherezova, J.M. Summy, A. GuapponeKoay, X. Shi, T. Mast, J. Pustula, H.G. Zot, N. Mazloum, M.Y. Lee, D.C. Flynn, Mol. Biol. Cell 13 (2002) 2311. [106] A. Gatesman, V.G. Walker, J.M. Baisden, S.A. Weed, D.C. Flynn, Mol. Cell. Biol. 24 (2004) 7578. [107] D.K. Werth, J.E. Niedel, I. Pastan, J. Biol. Chem. 258 (1983) 11423. [108] W.H. Ziegler, U. Tigges, A. Zieseniss, B.M. Jockusch, J. Biol. Chem. 277 (2002) 7396. [109] M. Perez-Moreno, A. Avila, S. Islas, S. Sanchez, L. GonzalezMariscal, J. Cell Sci. 111 (1998) 3563. [110] R. Massoumi, A. Sjo¨lander, J. Cell Sci. 114 (2001) 1925. [111] A. Woods, J.R. Couchman, J. Cell Sci. 101 (1992) 277. [112] J. Li, K.L. O’Connor, G.H. Greeley Jr., P.J. Blackshear, C.M. Townsend Jr., B.M. Evers, J. Biol. Chem. 280 (2005) 8351. [113] S. Barry, D. Critchley, J. Cell Sci. 107 (1994) 2033. [114] M. Holinstat, D. Mehta, T. Kozasa, R.D. Minshall, A.B. Malik, J. Biol. Chem. 278 (2003) 28793. [115] P.A. Janmey, U. Lindberg, Nat. Rev. Mol. Cell. Biol. 5 (2004) 658.
Review
Protein kinase C and the regulation of the actin cytoskeleton Christer Larsson Lund University, Dept of Laboratory Medicine, Molecular Medicine, Entrance 78, 3rd floor, UMAS SE-205 02, Malmo¨ University Hospital, Malmo¨, Sweden Received 27 June 2005; received in revised form 18 July 2005; accepted 18 July 2005 Available online 16 August 2005
Abstract Protein kinase C (PKC) isoforms are central components in intracellular networks that regulate a vast number of cellular processes. It has long been known that in most cell types, one or more PKC isoforms influences the morphology of the F-actin cytoskeleton and thereby regulates processes that are affected by remodelling of the microfilaments. These include cellular migration and neurite outgrowth. This review focuses on the role of classical and novel PKC isoforms in migration and neurite outgrowth, and highlights some regulatory steps that may be of importance in the regulation by PKC of migration and neurite outgrowth. Many studies indicate that integrins are crucial mediators both upstream and downstream of PKC in inducing morphological changes. Furthermore, a number of PKC substrates, directly associated with the microfilaments, such as MARCKS, GAP43, adducin, fascin, ERM proteins and others have been identified. Their potential role in PKC effects on the cytoskeleton is discussed. D 2005 Elsevier Inc. All rights reserved. Keywords: Protein kinase C; Actin; Integrins; Cell movement; Neurite; Stress fibers
Contents 1. 2.
Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Protein kinase C and its effects on the actin cytoskeleton . . . . . . . . . . . . . . . 2.1. Phorbol esters influence cellular morphology . . . . . . . . . . . . . . . . . . 2.2. PKC isoforms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Protein kinase C isoforms involved in the regulation of cell spreading and migration . 4. Protein kinase C and neurite outgrowth . . . . . . . . . . . . . . . . . . . . . . . . 5. Mechanisms of PKC effects on cellular morphology — integrins and other receptors. 6. Mechanisms of PKC effects on cellular morphology — PKC substrates. . . . . . . . 6.1. MARCKS and GAP43 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2. Adducin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3. Fascin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.4. ERM proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.5. AFAP-110 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.6. Vinculin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.7. Summary of PKC substrates . . . . . . . . . . . . . . . . . . . . . . . . . . 7. PKC induction of stress fibres . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
E-mail address: [email protected]. 0898-6568/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.cellsig.2005.07.010
. . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . .
277 277 277 277 278 278 279 279 280 280 280 281 281 282 282 282 282 282 282
C. Larsson / Cellular Signalling 18 (2006) 276 – 284
1. Introduction Many morphological changes of a cell are at least partially driven by a remodelling of the actin cytoskeleton. There are many instances when it would be beneficial to be able to influence these changes. One example of detrimental effects of altered regulation of these processes is when a cell has acquired the capacity to migrate and to invade a tissue which is a prerequisite for a full-blown malignant phenotype of a cancer cell. In this case, it would be of benefit to be able to suppress the cytoskeletal activity. On the other hand, in some instances a possibility to facilitate the cytoskeletal processes would be advantageous. One such example concerns damaged axons in the central nervous system, which cannot regenerate spontaneously. A better understanding of the regulation of the cytoskeleton would thus facilitate our search for treatments for many diseases.
2. Protein kinase C and its effects on the actin cytoskeleton 2.1. Phorbol esters influence cellular morphology The morphology of the cytoskeleton is regulated by a large number of components and can be modified by many exogenous stimuli. It has for a long time been known that phorbol esters can stimulate or enhance cellular migration and motility [1 –6]. This is likely related to the more immediate effects on the actin cytoskeleton that also are observed upon addition of phorbol esters. These include cells spreading and ruffling [3,7] and dismantling of stress fibres [7– 9], although in some cell types stress fibres are formed upon phorbol ester exposure [10]. Phorbol esters are well-known activators of classical and novel protein kinase C (PKC) isoforms and it can therefore be assumed that one or several of these PKC isoforms promote changes in the cytoskeleton that facilitate or drive cell spreading and migration. However, phorbol esters bind and activate several other proteins that contain typical C1 domains [11] and are putative regulators of the microfilament morphology. These include the chimaerins which are GTPase-activating proteins and downregulate the activity of the Rac GTPase [12,13], the myotonic dystrophy kinaserelated Cdc42-binding kinase (MRCK) [14 – 16] and RasGRPs [17,18]. It is therefore likely that some phorbol ester-induced effects are mediated via one or several of these proteins. Nevertheless, the use of PKC inhibitors and other tools have clearly indicated that classical and novel PKC isoforms have significant roles in regulating cytoskeletondriven processes in cells. 2.2. PKC isoforms The PKC isoforms constitute a family of, depending on classification criteria, 10– 15 members [19]. In general,
277
classical (PKCa, hI, hII and g), novel (PKCy, q, D and u) and atypical (PKCN/E and ~) isoforms are considered as ‘‘proper’’ PKCs. Only classical and novel PKC isoforms contain C1 domains that bind phorbol esters and diacylglycerol (DAG). This review focuses on the role of classical and novel isoforms in the regulation of morphological changes driven by alterations in the F-actin structure. All PKC isoforms consist of one regulatory and one catalytic domain, with the regulatory domain containing binding sites for the PKC activators. The basic structure of the classical and novel PKCs is depicted in Fig. 1. Briefly, the regulatory domain of classical isoforms contains one Ca2+-binding C2 domain and two C1 domains which can bind DAG and phorbol esters. The general view of PKC activation has been that when the levels of Ca2+ and/or DAG increase in the cell they bind the C2 and C1 domains respectively, recruit the classical PKC to the plasma membrane and induce a conformational change of the enzyme. The kinase domain is thus exposed and can exert its catalytic function. A similar general mechanism is thought to operate for novel isoforms, with the exception that they are insensitive to Ca2+. Many studies have also shown that beside activation of PKC by membrane association, PKC is also regulated by interaction with other proteins, which will through interaction either modify the conformation of PKC or stabilise the activated conformation. The regulation of PKC has been reviewed in many papers, see for instance [20 – 22]. It is clear that the different isoforms have unique effects in cellular regulation. During the last 10 –15 years we have begun to understand how the different isoforms uniquely regulate specific cellular processes. This has been possible by using different approaches such as overexpression of isoforms, inhibitors with isoform specificity such as Go¨6976 and rottlerin, expression of dominant negative variants, cells with targeted deletion of one specific isoform or introduction of peptides that interfere with inhibitory intramolecular interactions or with interactions of PKC with other proteins. All these approaches have their advantages but also their limitations. The specificity of the inhibitors is clearly questionable, and the similarity between isoforms makes expression of large amounts of wild type PKCs, peptides, or Classical PKCs (α,βΙ,βΙΙ,γ) PS
C1a
C1b
C2
kinase
V5
Novel PKCs (δ,ε,η,θ) C2
PS
C1a
C1b
kinase
V5
Fig. 1. The overall structure of classical and novel PKC isoforms. The PKC isoforms consists of an N-terminal regulatory domain containing a pseudosubstrate (PS) two C1 domains (C1a and C1b) and one C2 domain and a C-terminal kinase domain with a V5 region that displays large variability between the isoforms. The C2 domain in classical isoforms binds Ca2+, whereas the same domain in novel isoforms does not bind Ca2+.
278
C. Larsson / Cellular Signalling 18 (2006) 276 – 284
dominant negative variants, not easy to interpret, due to the risk of unspecific effects. This can be at least partially circumvented by expressing analogous variants of several isoforms. Due to the limitations of the techniques some data on the importance of a certain isoform may therefore actually demonstrate what an isoform can do, rather than what an isoform really does.
3. Protein kinase C isoforms involved in the regulation of cell spreading and migration PKCa is perhaps the isoform that has emerged as a general promoter of cell spreading and migration. Elevating the levels of PKCa promotes migration of endothelial cells [23], MCF7 breast carcinoma cells [24] and 2C4 fibrosarcoma cells [25] and spreading of CHO cells [26] and PKCa levels correlate with the migratory capacity of colon carcinoma cells [27]. Furthermore, suppression of PKCa either by antisense approaches [28,29] or by dominant negative approaches [30] leads to impaired migration of vascular smooth muscle cells, endothelial cells and melanoma cells. However there are also indications that other isoforms promote migration. Downregulation or gene ablation of PKCh suppresses the migration of T cells on LFA-1 ligand [31] and MCP-1-stimulated chemotaxis of human monocytes [32]; and PKChII conceivably mediates the glucose potentiation of PDGF-stimulated migration of vascular smooth muscle cells [33] and PKChI contributes to cell migration on fibrinogen [34]. PKCy is important for stressinduced smooth muscle cell migration [35] and EGFstimulated motility of fibroblasts [36] and the levels of PKCy correlates to the metastatic potential of rat mammary carcinoma cell lines [37]. PKCq has been suggested to promote glioma cell migration [38] and it is necessary for hepatocyte growth factor-stimulated motility [39] and for cellular movement on fibronectin [40]. In addition, activated PKCq can rescue cell spreading in cells devoid of h1 integrin signalling [41]. There is also evidence for a role for PKCu. Kinase-inactive PKCu suppresses capillary endothelial cell migration [42]. Thus, a general effect of PKC in many cell types is to promote migration. In particular PKCa has this capacity but the relative contribution of different isoforms in vivo still needs to be firmly established.
4. Protein kinase C and neurite outgrowth The outgrowth of neurites, immature processes from neuronal cells that later may mature into axons or dendrites, is a crucial process for the differentiation of neurons. The outgrowth is led by the growth cone, a highly dynamic structure on the tip of the growing neurite. The morphology of the growth cone seems to be formed by similar changes in the F-actin structure as those that is seen in the spreading and migrating cell.
As for spreading and migration, phorbol esters have for a long time been known to have both positive and negative effects on neurite outgrowth. Initially it was seen that phorbol esters suppress neurite outgrowth from mouse neuroblastoma cells [43] and embryonic chick ganglia [44]. However, later studies have shown that the major effect may be to induce neurites exemplified by the effect on cultured human neuroblastoma cells [45], chick embryo sensory, sympathetic and parasympathetic ganglion cells [46,47], cerebellar granule cells [48] and on primary neurons cultured either on extracellular matrix proteins or on cadherins [49,50]. There are many studies indicating important roles for novel PKC isoforms in promoting neurite outgrowth. In PC12 cells, phorbol esters by themselves do not induce neurites but they potentiate neurite outgrowth induced by nerve growth factor (NGF), which is correlated to a translocation of PKCy [51] and PKCy has been suggested to mediated the activation of MAP kinases in PC12 upon neurogenic, but not mitogenic, stimulation [52]. On the other hand, increased levels of PKCq but not of PKCy potentiates neurite outgrowth induced by NGF or epidermal growth factor (EGF) from the same cells [53,54] and by itself induces neurites in cultured human neuroblastoma cells [55] and in immortalised neural precursor cells from the hippocampus and medullary raphe [56]. The latter effects are independent of the catalytic activity of PKC and mediated by the C1 domains. There is a motif N-terminal of the C1b domain that is conserved in all novel isoforms that is crucial for the neurite-inducing effect of PKC [57] suggesting that all novel isoforms may have the potential to induce neurites via this mechanism. Also classical isoforms may be of importance for some neurite-inducing stimuli such as the NCAM-mediated outgrowth of neurites [58,59]. The above mentioned studies describe more long term effects on the morphology of neuronal cells. When more local immediate effects on the growth cone are studied, PKC frequently turns out to mediate signals that either induce a collapse of the growth cone or have a repellent effect on its growth. Dopamine induces retraction of retinal horizontal cell neurites which seems to be mediated via PKC and can be mimicked by PKC activators [60] and the PTPA- and thrombin-induced growth cone collapse is conceivably transduced via PKCy [61] and PKCq [62] respectively and PKC inhibitors suppresses the repellent effects of myelin components on neurite outgrowth [63]. Furthermore, the CXCR4-induced repulsion of growth cones of both cerebellar granule cells and Xenopus spinal neurons are dependent on a classical PKC isoform [64]. Thus, a large number of studies indicate a general promoting effect in a long term perspective whereas PKC generally seems to, in the short term perspective, mediate growth cone collapse and retraction. These seemingly conflicting results may be due to the same protein having different short term and long term effects. Alternatively it
C. Larsson / Cellular Signalling 18 (2006) 276 – 284
may be related to the importance of different isoforms for the effects. The fact that PKC may mediate some effects on neurites via its regulatory domain further complicates the interpretation of the data.
5. Mechanisms of PKC effects on cellular morphology — integrins and other receptors The fact that there is no clear common effect of PKC isoforms for different cell types indicates that PKC may mediate its effects via several mechanisms. Such a conclusion is supported by the literature which indicates a wide range of pathways that may mediate the PKC effect, including direct phosphorylation of cytoskeletal regulators, influence on other signalling pathways and effects on receptors for extracellular matrix component. In this review the focus is on substrates directly involved in the modification of the actin cytoskeleton and on receptors of components of the extracellular matrix. Integrins constitute an important pool of the surface molecules that mediate the binding of the cell with the extracellular matrix. This interaction with the surrounding tissue not only acts as a structural element but also conveys signals into the cell. In order for a cell to move, there needs to be a constant replenishment of integrins to the front of the cell at the same time as integrins at the rear or at desmosomes need to be detached. There are now a large amount of studies that demonstrate an important role for PKC in the regulation of integrins and in the transduction of integrin signals. There are many reports of association and co-localisation of PKC isoforms with integrins. It is difficult to a discern a pattern of isoform-specific effects but many studies indicate that in particular PKCa [24,65,66] and/or PKCq [38,40] associates with h1 integrin. On the other hand, for h3 integrin, PKCh has been shown to be an important interacting partner [34]. For some of the interactions the binding may be indirect and dependent on RACK1 [34,38]. There is now considerable evidence that an important function of PKC is to regulate the transport and distribution of integrins. Increasing the levels of PKCa leads to more h1 integrins on the cellular surface and following PKC activation there is an internalisation of the integrins [24]. PKCa is also important for clustering-induced internalisation of a2/h1 integrins [67] and necessary for ligand induced internalisation of c-met, indicating that internalisation of receptors may be a common effect for PKCa. The internalisation of c-met is important for the activation of several transduction cascades and for cellular migration [39]. However, PKCa activity is not only important for the internalisation of integrins. PKCa-induced relocation of integrins often results in more integrins at the leading edge, which is in line with PKC having a pro-migratory effect. 12o-tetradecanoyl phorbol 13-acetate (TPA) stimulates the movement of PKC vesicles containing PKCa and h1
279
integrins to the leading edge, and disruption of the PKCa interaction with h1 integrins suppresses both TPA and EGFstimulated migration [65]. a6/h4 integrins are also targets for PKC. TPA induces transport of a6/h4 with tetraspanin 6.1 to the leading edge in pancreatic carcinoma [68] and it has been shown that PKCa phosphorylates h4 integrin [69] and leads to a relocation of a6/h4 from hemidesmosomes to the leading edge [70] during both TPA- and EGF-stimulated migration of A431 cells. The PKC-mediated translocation of integrins can be mediated by other receptors for extracellular matrix components. One cell surface receptor that is of particular interest in this aspect is syndecan-4. This is a proteoglycan that binds several extracellular matrix proteins and upon ligation in the presence of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] activates PKCa. Binding to syndecan-4 is actually sufficient to induce a h1 integrin-dependent cell spreading and stress fibre formation. This is mediated via PKCa [26]. The adhesion of cells to integrin ligands frequently makes the cell spread and in many instances it also makes the cell migrate. It is conceivable that PKC in a lot of these cases is crucial for the transduction of the signal from integrin ligation to cell spreading. T-cells devoid of PKCh do not migrate on LFA-1 ligand and migration can be rescued by expression of PKChI [31] and spreading of PKCh null platelets on fibrinogen is suppressed compared to wild type platelets [34]. Furthermore, PKCq null fibroblasts have impaired migration on fibronectin [40]. PKC is not only crucial for integrin-mediated effects in some cell types. Activation of PKC can actually rescue some characteristics that are lost in cells in which integrin signalling has been abrogated. PKCq rescues cell spreading on fibrinogen and collagen I of cells in which signalling through h1 integrins had been destroyed [41] and activation of either PKCa, PKCy or PKCq rescues spreading of a5 integrin-deficient cells on fibronectin [71]. In some cases, PKC complements integrin ligation and potentiates the morphological effects that is dependent on or mediated via integrins. For melanoma cells, binding to fibronectin via a5h1 integrins leads to spreading and further stimulation of syndecan-4 activates PKCa with the formation of focal adhesions subsequent cellular migration as result. However, migration and formation of focal adhesions, stimulated by spreading on a4h1 integrin ligands are independent of PKC [30]. An effect along the same line is seen in CHO cells expressing a2bh3 integrins. These cells weakly adhere to antibodies, but addition of TPA makes the cell spread [4].
6. Mechanisms of PKC effects on cellular morphology — PKC substrates It seems clear that modification of integrin function, control of integrin localisation and transduction of integrin
280
C. Larsson / Cellular Signalling 18 (2006) 276 – 284
signals seems to be important mechanisms for PKC effects on the cytoskeleton. Other components that are conceivably central in the mediation of the PKC effects are the substrates of PKC that have been shown to directly influence the morphology of the actin cytoskeleton. There are a number of such substrates described; some were identified more than twenty years ago whereas some were recently characterised. Here some of them are discussed. 6.1. MARCKS and GAP43 This family of related proteins consisting of MARCKS, MacMARCKS, GAP43 and CAP23 have long been candidates for mediating PKC effects on the cytoskeleton. MARCKS was one of the first PKC substrates that was characterised. PKC-mediated phosphorylation of an 87 kD protein was described already in 1982 [72] and the protein was later identified as MARCKS [73]. MacMARCKS, a closely related protein, was isolated shortly thereafter [74,75]. MARCKS is a membrane-bound protein. The membrane interaction is dependent on both an N-terminal myristoylation of MARCKS and on a cluster of basic residues in its effector domain. The myristic acid is inserted into membranes and the basic residues interact with negatively charged head groups of membrane phospholipids. The PKC phosphorylation sites are located in the effector domain and phosphorylation alters the net charge of the domain, which results in a dissociation of MARCKS from the membrane [76]. MARCKS phosphorylation can be carried out by both classical and novel PKC isoforms [77]. GAP43 is an analogous protein with an effector domain and N-terminal palmitoylation. It was initially detected as a phosphoprotein in synaptosomes [78] and independently as a substrate of PKC, the phosphorylation of which correlated to synaptic plasticity [79]. Depletion of GAP43 markedly alters neurite and growth cone morphology [80] and overexpression of GAP43 in the nervous system promotes neurite sprouting, an effect that is dependent on its PKC phosphorylation sites [81]. As for MARCKS, GAP43 can be phosphorylated by classical and novel isoforms [82]. MARCKS has been suggested to be an important factor for the anchoring of the actin cytoskeleton to the plasma membrane [83]. A dissociation of MARCKS from the membrane would thereby lead to a detachment of microfilaments from the membrane. However, there are later indications that MARCKS exerts its functions by sequestering PI(4,5)P2 [84,85] and a release of MARCKS from the membrane would then lead to an increase in available PI(4,5)P2. This mechanism appears to be common for MARCKS, CAP23 and GAP43 and primarily takes place at rafts rich in PI(4,5)P2 [86]. GAP43, in a manner dependent on its phosphorylation status has also been shown to affect the structure of actin filaments with the phosphorylated variant promoting the stabilisation of long filaments [87]. There are clear indications that phosphorylation of MARCKS proteins are important for some PKC effects on
the cytoskeleton. Dominant negative, i.e. non-phosphorylatable variants, MacMARCKS [88] and MARCKS [89] suppress phorbol ester-mediated cell spreading. It seems like a dynamic relocation of MARCKS is necessary for the spreading and subsequent formation of focal adhesions in myoblasts seeded on fibronectin. During spreading there is a PKC-dependent phosphorylation and translocation to the cytosol, whereas later MARCKS translocates to the focal adhesions that are under assembly [71,90].This may thus reflect a role for MARCKS in regulating the local F-actin structure. PKC phosphorylation of MacMARCKS has been shown to be important for TPA-mediated lateral diffusion of h2 integrins [91], suggesting that MacMARCKS may also anchor integrins. A plausible effect of PKC phosphorylation of MARCKS and GAP43 is thus an increased availability of PI(4,5)P2 in the plasma membrane which can result in a dynamic reorganisation of the cortical microfilaments. However, the PKC effect is conceivably not limited to an ‘‘inactivation’’ of the sequestering of PI(4,5)P2. There are, as mentioned above, experimental evidence demonstrating that PKC phosphorylation must give also other functions to these proteins, than merely ablate their binding to PI(4,5)P2. 6.2. Adducin Adducin is a protein that simultaneously caps F-actin and assembles F-actin with spectrin, a function that is fundamental for the shape of the erythrocyte [92]. PKC phosphorylates adducin from erythrocytes [93], brain [94] and fibroblasts [95], which leads to a diminished activity of adducin in terms of both F-actin-capping and promotion of spectrin-binding to F-actin [96]. A PKC-mediated loss of adducin interactions with F-actin and spectrin, resulting in exposure of free barbed ends, is conceivably important for the PKC-mediated morphological changes that take place during platelet activation [97]. Phosphorylated adducin is also found in dendritic spines, which are dynamic structures, indicating a role in cytoskeletal remodelling [96]. 6.3. Fascin Fascin is a protein that tightly bundles F-actin and is important for the formation of actin-based protrusions and for maintaining cytoplasmic F-actin bundles. The bundling activity of fascin is dependent on its capacity to crosslink actin monomers and this is inhibited by phosphorylation of S39 [98]. The phosphorylation status of this site is dependent on the extracellular matrix. When C2C12 myoblasts are cultured on thrombospondin-1 the site is dephosphorylated but seeding onto fibronectin induces a PKC-mediated phosphorylation of S39 [99]. The phosphorylation is conceivably mediated by PKCa [100] and it leads to the dissociation of fascin from the F-actin bundles and a diffuse localisation of fascin. This is likely of importance for cell spreading since a S39A fascin mutant
C. Larsson / Cellular Signalling 18 (2006) 276 – 284
suppresses spreading on fibronectin whereas a S39D mutant promotes it [99]. However, the role of the PKCa-fascin connection seems to be more elusive. PKCa can directly bind phosphorylated fascin and disruption of this interaction suppresses MCF-7 cell spreading but actually potentiates the cell migration on fibronectin and the formation of focal adhesions [100]. Thus, when seeded on fibronectin PKCa phosphorylates fascin which dissociates from F-actin and thereby releases the tight bundles. This is important for optimal spreading. PKCa binds the phosphorylated fascin and the complex is important for fine-tuning of the migratory response. It will be of interest to know if fascin targets PKC to other substrates or whether the main function of the persistent interaction is to maintain fascin in a phosphorylated state. 6.4. ERM proteins The ERM (ezrin-radixin-moesin) proteins constitute an interesting group of proteins that both function as connectors of the microfilaments with the plasma membrane as well as transduce signals to different transduction pathways. ERM proteins are maintained in a dormant state by an intramolecular interaction between the N-terminal FERM domain and the C-terminus. This interaction, with a subsequent conformational release of the protein, can be disrupted by phosphorylation of a threonine residue in the C-terminus [101]. For moesin, PKCu has been shown to phosphorylate this threonine (T558) residue [102] with a consequent exposure of both the N- and C-termini and increased interaction with cortical microfilaments [103]. In addition, PKCa has been shown to bind to and phosphorylate the corresponding residue in ezrin. The proteins colocalise in the leading edge of migrating cells and the phosphorylation is likely of functional importance since overexpression of T558A ezrin mutant suppresses migration in PKCa-overexpressing cells [25]. The activation of selected ERM proteins may therefore be an important pathway in mediating PKC effects on migration. The ERM proteins also interact with adhesion molecules including CD44 and ICAMs [101]. This may be another site for PKC action. PKC activation leads to a dephosphorylation of S325 at same time as it phosphorylates S291 in CD44. This alteration of phosphorylation decreases the interaction of CD44 with ezrin and a S291A mutant suppresses TPA-induced migration [104]. Thus, PKC can influence ERM protein activities both by altering their conformation and by determining the binding capacity of their interaction partners. 6.5. AFAP-110 AFAP-110 is a multi-domain protein that may convey some PKC signals to the cytoskeleton. AFAP-110 can both cross-link F-actin and bind Src which results in the activation of Src. Classical PKCs bind to AFAP-110 and
281
phosphorylates it and disrupts its multimerisation [105] and makes it able to activate Src [106]. Expression of an AFAP110 mutant with deleted PKC-binding site suppresses TPAinduced ruffling indicating a role for AFAP-110 in the TPA effect [105]. AFAP-110 also seems to be important for the formation of podosomes [106]. It remains to be seen if all AFAP-110-mediated effects are due to the subsequent MARCKS/GAP43 PKC
Adducin PKC
Fascin PKC
ERM proteins PKC
Fig. 2. A rough sketch of the effects of PKC phosphorylation on the function of some PKC substrates. PKC substrates are depicted in green, Factin in red, PI(4,5)P2 in blue, other proteins in orange and yellow and the plasma membrane is black. MARCKS/GAP43: The unphosphorylated protein sequesters PI(4,5)P2 and may also couple F-actin to the plasma membrane. These functions are lost upon phosphorylation. The free PI(4,5)P2 will recruit actin-modifying proteins and the net effect will be to increase actin assembly [115]. Adducin: Adducin caps the barbed end of F-actin and supports the interaction of F-actin with spectrin. A phosphorylation by PKC results in a dissociation of the complex and the exposure of free barbed ends on which actin polymerisation can take place. Fascin: Fascin bundles F-actin in tight structures. The binding to actin is lost upon PKC phosphorylation of fascin leading to the dissociation of the bundles. ERM proteins: In the resting state, these proteins are localised in the cytosol and kept in a closed conformation by an intramolecular interaction between the N-terminal FERM domain and the C-terminal part of the protein. PKC phosphorylation disrupts this interaction and opens up the conformation. The exposed FERM domain interacts with plasma membrane proteins and the C-terminus can bind F-actin. In the open conformation, ERM proteins can interact with many signalling proteins and thereby focus further signalling to the plasma membrane.
282
C. Larsson / Cellular Signalling 18 (2006) 276 – 284
activation of Src or if any of them are related to direct effects on the F-actin structure.
to the RhoA pathway. PKCa has been shown to mediate thrombin-induced activation of p115RhoGEF, an activator of RhoA, and induction of stress fibres [114].
6.6. Vinculin Vinculin is a PKC substrate [107] and PKCa has been shown to bind to and phosphorylate vinculin [108]. This may explain why in some cell systems the translocation of vinculin to focal adhesions and the formation of different cellular adhesions are dependent on PKC [109], in particular on PKCa [110]. 6.7. Summary of PKC substrates Several of the above listed substrates may contribute to PKC-induced spreading and migration. These events require a modification of the cortical cytoskeleton and phosphorylation of MARCKS and/or GAP43 will cause the release of PI(4,5)P2 and thereby a local accumulation of F-actin modifying proteins. Phosphorylation of adducin will lead to a dissociation of spectrin from F-actin and exposure of free barbed ends. PKC activation will likewise make fascin loose its bundling activity leading to a less rigid F-actin structure and ERM proteins may be recruited to plasma membrane proteins and interact with F-actin or change the activity of other signalling proteins. Thus, there are several mechanisms through which a local activation of PKC may lead to dynamic changes in the local cortical cytoskeleton. These events are summarised in Fig. 2.
8. Conclusions It is clear that PKC regulates the actin cytoskeleton in a wide range of cell types and a general activation of PKC isoforms leads to suppression of stress fibres, formation of ruffles and a pro-migratory effect in most cells that have been studied. However, PKC activation can also lead to formation of stress fibres in some cell types. This may be related to specific and different effects of the PKC isoforms. A general impression of the literature is that particularly PKCa has the capacity to alter the cytoskeleton in a promigratory manner and to promote migration and that PKCy is involved in the RhoA pathway. This may be due to the fact that primarily these isoforms have been studied and that other PKCs could have the same effects in the experimental systems. It will be of interest to see which isoforms that mediate the effects in vivo. A large number of PKC substrates have been identified and are conceivable candidates for mediating the PKC effects on the cytoskeleton. However, there are undoubtedly substrates that remain to be characterised and also interacting proteins that are of importance for the PKC effects.
Acknowledgements 7. PKC induction of stress fibres As discussed above, in most cell types a general PKC activation leads to a loss of stress fibres and induction of ruffles and an increased tendency to migration. However, there are also cases when PKC seems to promote or drive the formation of stress fibres. The formation of focal adhesions and stress fibres in fibroblasts that have spread on fibronectin is dependent on and can be induced by PKC [111]. The leukotrine-induced stress fibre formation in intestinal epithelial cells is for instance mediated by PKC, presumably PKCy, and the effect can be obtained by a direct activation of PKC [10]. Leukotriene-stimulated PKC activation seems to be mediated by RhoA and there are cases when PKCy is downstream of RhoA or mimicks RhoA activation. Rho/ ROCK-mediated MARCKS phosphorylation in endocrine cells is dependent on PKCy [112] and PKCy activation is crucial for EGF-induced phosphorylation of the myosin light chain in fibroblasts [36]. The facts that overexpression of PKCy in endothelial cells make them more prone to form focal adhesion and suppresses their migratory tendency, the opposite to what is observed with PKCa [23], and that RhoA induces focal adhesions that contain PPKCy [113] also supports a role for PKCy in mediating or inducing RhoA effects. However, it is not only PKCy that has been coupled
This work is supported by the Swedish Cancer Society, the Swedish Research Council, the Swedish Children’s Cancer Foundation, Malmo¨ University Hospital Research funds and the Crafoord, Greta and Johan Kock, and Ollie and Elof Ericsson Foundations.
References [1] [2] [3] [4]
[5] [6] [7] [8] [9] [10] [11] [12]
J. Varani, S.E. Fligiel, P. Perone, Int. J. Cancer 35 (1985) 559. R. Sears, G. Ciment, Dev. Biol. 130 (1988) 133. K. Vuori, E. Ruoslahti, J. Biol. Chem. 268 (1993) 21459. P. Defilippi, M. Venturino, D. Gulino, A. Duperray, P. Boquet, C. Fiorentini, G. Volpe, M. Palmieri, L. Silengo, G. Tarone, J. Biol. Chem. 272 (1997) 21726. F. Entschladen, B. Niggemann, K. Zanker, P. Friedl, J. Immunol. 159 (1997) 3203. V. Rigot, M. Lehmann, F. Andre, N. Daemi, J. Marvaldi, J. Luis, J. Cell Sci. 111 (1998) 3119. M. Schliwa, T. Nakamura, K.R. Porter, U. Euteneuer, J. Cell Biol. 99 (1984) 1045. A.J. Ridley, A. Hall, EMBO J. 13 (1994) 2600. D. Brandt, M. Gimona, M. Hillmann, H. Haller, H. Mischak, J. Biol. Chem. 277 (2002) 20903. R. Massoumi, C. Larsson, A. Sjo¨lander, J. Cell Sci. 115 (2002) 3509. D. Ron, M.G. Kazanietz, FASEB J. 13 (1999) 1658. S. Ahmed, R. Kozma, C. Monfries, C. Hall, H.H. Lim, P. Smith, L. Lim, Biochem. J. 272 (1990) 767.
C. Larsson / Cellular Signalling 18 (2006) 276 – 284 [13] R. Kozma, S. Ahmed, A. Best, L. Lim, Mol. Cell Biol. 16 (1996) 5069. [14] T. Leung, X.Q. Chen, I. Tan, E. Manser, L. Lim, Mol. Cell Biol. 18 (1998) 130. [15] I. Tan, K.T. Seow, L. Lim, T. Leung, Mol. Cell. Biol. 21 (2001) 2767. [16] S. Wilkinson, H.F. Paterson, C.J. Marshall, Nat. Cell Biol. 7 (2005) 255. [17] J.O. Ebinu, D.A. Bottorff, E.Y. Chan, S.L. Stang, R.J. Dunn, J.C. Stone, Science 280 (1998) 1082. [18] C.E. Tognon, H.E. Kirk, L.A. Passmore, I.P. Whitehead, C.J. Der, R.J. Kay, Mol. Cell Biol. 18 (1998) 6995. [19] H. Mellor, P.J. Parker, Biochem. J. 332 (1998) 281. [20] A.C. Newton, Chem. Rev. 101 (2001) 2353. [21] D. Schechtman, D. Mochly-Rosen, Oncogene 20 (2001) 6339. [22] Y. Shirai, N. Saito, J. Biochem. (Tokyo) 132 (2002) 663. [23] E.O. Harrington, J. Loffler, P.R. Nelson, K.C. Kent, M. Simons, J.A. Ware, J. Biol. Chem. 272 (1997) 7390. [24] T. Ng, D. Shima, A. Squire, P.I.H. Bastiaens, S. Gschmeissner, M.J. Humphries, P.J. Parker, EMBO J. 18 (1999) 3909. [25] T. Ng, M. Parsons, W.E. Hughes, J. Monypenny, D. Zicha, A. Gautreau, M. Arpin, S. Gschmeissner, P.J. Verveer, P.I.H. Bastiaens, P.J. Parker, EMBO J. 20 (2001) 2723. [26] C.K. Thodeti, R. Albrechtsen, M. Grauslund, M. Asmar, C. Larsson, Y. Takada, A.M. Mercurio, J.R. Couchman, U.M. Wewer, J. Biol. Chem. 278 (2003) 9576. [27] K. Masur, K. Lang, B. Niggemann, K.S. Zanker, F. Entschladen, Mol. Biol. Cell 12 (2001) 1973. [28] H. Haller, C. Lindschau, C. Maasch, H. Olthoff, D. Kurscheid, F.C. Luft, Circ. Res. 82 (1998) 157. [29] A. Wang, M. Nomura, S. Patan, J.A. Ware, Circ. Res. 90 (2002) 609. [30] Z. Mostafavi-Pour, J.A. Askari, S.J. Parkinson, P.J. Parker, T.T.C. Ng, M.J. Humphries, J. Cell Biol. 161 (2003) 155. [31] Y. Volkov, A. Long, S. McGrath, D. Ni Eidhin, D. Kelleher, Nat. Immunol. 2 (2001) 508. [32] K.A. Carnevale, M.K. Cathcart, J. Biol. Chem. 278 (2003) 25317. [33] M. Campbell, E.R. Trimble, Circ. Res. 96 (2005) 197. [34] C.S. Buensuceso, A. Obergfell, A. Soriani, K. Eto, W.B. Kiosses, E.G. Arias-Salgado, T. Kawakami, S.J. Shattil, J. Biol. Chem. 280 (2005) 644. [35] C. Li, F. Wernig, M. Leitges, Y. Hu, Q. Xu, FASEB J. 17 (2003) 2106. [36] A. Iwabu, K. Smith, F.D. Allen, D.A. Lauffenburger, A. Wells, J. Biol. Chem. 279 (2004) 14551. [37] S.C. Kiley, K.J. Clark, S.K. Duddy, D.R. Welch, S. Jaken, Oncogene 18 (1999) 6748. [38] A. Besson, T.L. Wilson, V.W. Yong, J. Biol. Chem. 277 (2002) 22073. [39] S. Kermorgant, D. Zicha, P.J. Parker, EMBO J. 23 (2004) 3721. [40] J. Ivaska, R.D.H. Whelan, R. Watson, P.J. Parker, EMBO J. 21 (2002) 3608. [41] A.L. Berrier, A.M. Mastrangelo, J. Downward, M. Ginsberg, S.E. LaFlamme, J. Cell Biol. 151 (2000) 1549. [42] S. Tang, K.G. Morgan, C. Parker, J.A. Ware, J. Biol. Chem. 272 (1997) 28704. [43] D.N. Ishii, Cancer Res. 38 (1978) 3886. [44] D.N. Ishii, E. Fibach, H. Yamasaki, I.B. Weinstein, Science 200 (1978) 556. [45] S. Pa˚hlman, L. Odelstad, E. Larsson, G. Grotte, K. Nilsson, Int. J. Cancer 28 (1981) 583. [46] L. Hsu, D. Natyzak, J.D. Laskin, Cancer Res. 44 (1984) 4607. [47] L. Hsu, Neurosci. Lett. 62 (1985) 283. [48] M.A. Cambray-Deakin, J. Adu, R.D. Burgoyne, Brain Res. Dev. Brain Res. 53 (1990) 40. [49] J.L. Bixby, P. Jhabvala, J. Cell Biol. 111 (1990) 2725. [50] J.L. Bixby, Neuron 3 (1989) 287. [51] K.R. O’Driscoll, K.K. Teng, D. Fabbro, L.A. Greene, I.B. Weinstein, Mol. Cell. Biol. 6 (1995) 449.
283
[52] K.C. Corbit, D.A. Foster, M.R. Rosner, Mol. Cell. Biol. 19 (1999) 4209. [53] B. Hundle, T. McMahon, J. Dadgar, R.O. Messing, J. Biol. Chem. 270 (1995) 30134. [54] C. Brodie, K. Bogi, P. Acs, P. Lazarovici, G. Petrovics, W.B. Anderson, P.M. Blumberg, Cell Growth Differ. 10 (1999) 183. [55] R. Zeidman, B. Lo¨fgren, S. Pa˚hlman, C. Larsson, J. Cell Biol. 145 (1999) 713. [56] M. Ling, U. Trolle´r, R. Zeidman, C. Lundberg, C. Larsson, Exp. Cell Res. 292 (2004) 135. [57] M. Ling, U. Troller, R. Zeidman, H. Stensman, A. Schultz, C. Larsson, J. Biol. Chem. 280 (2005) 17910. [58] I. Leshchyns’ka, V. Sytnyk, J.S. Morrow, M. Schachner, J. Cell Biol. 161 (2003) 625. [59] K. Kolkova, H. Stensman, V. Berezin, E. Bock, C. Larsson, J. Neurochem. 92 (2005) 886. [60] S. Rodrigues Pdos, J.E. Dowling, Proc. Natl. Acad. Sci. U. S. A. 87 (1990) 9693. [61] S.E. Ensslen, S.M. Brady-Kalnay, Mol. Cell. Neurosci. 25 (2004) 558. [62] K. Mikule, S. Sunpaweravong, J.C. Gatlin, K.H. Pfenninger, J. Biol. Chem. 278 (2003) 21168. [63] R. Sivasankaran, J. Pei, K.C. Wang, Y.P. Zhang, C.B. Shields, X.M. Xu, Z. He, Nat. Neurosci. 7 (2004) 261. [64] Y. Xiang, Y. Li, Z. Zhang, K. Cui, S. Wang, X.B. Yuan, C.P. Wu, M.M. Poo, S. Duan, Nat. Neurosci. 5 (2002) 843. [65] M. Parsons, M.D. Keppler, A. Kline, A. Messent, M.J. Humphries, R. Gilchrist, I.R. Hart, C. Quittau-Prevostel, W.E. Hughes, P.J. Parker, T. Ng, Mol. Cell. Biol. 22 (2002) 5897. [66] K. Podar, Y.-T. Tai, B.K. Lin, R.P. Narsimhan, M. Sattler, T. Kijima, R. Salgia, D. Gupta, D. Chauhan, K.C. Anderson, J. Biol. Chem. 277 (2002) 7875. [67] P. Upla, V. Marjomaki, P. Kankaanpaa, J. Ivaska, T. Hyypia, F.G. van der Goot, J. Heino, Mol. Biol. Cell 15 (2004) 625. [68] M. Herlevsen, D.-S. Schmidt, K. Miyazaki, M.J. Zoller, Cell Sci. 116 (2003) 4373. [69] I. Rabinovitz, L. Tsomo, A.M. Mercurio, Mol. Cell. Biol. 24 (2004) 4351. [70] I. Rabinovitz, A. Toker, A.M. Mercurio, J. Cell Biol. 146 (1999) 1147. [71] M.-H. Disatnik, S.C. Boutet, C.H. Lee, D. Mochly-Rosen, T.A. Rando, J. Cell Sci. 115 (2002) 2151. [72] W.C. Wu, S.I. Walaas, A.C. Nairn, P. Greengard, Proc. Natl. Acad. Sci. U. S. A. 79 (1982) 5249. [73] D.J. Stumpo, J.M. Graff, K.A. Albert, P. Greengard, P.J. Blackshear, Proc. Natl. Acad. Sci. U. S. A. 86 (1989) 4012. [74] P.J. Blackshear, G.M. Verghese, J.D. Johnson, D.M. Haupt, D.J. Stumpo, J. Biol. Chem. 267 (1992) 13540. [75] J. Li, A. Aderem, Cell 70 (1992) 791. [76] M. Thelen, A. Rosen, A.C. Nairn, A. Aderem, Nature 351 (1991) 320. [77] A. Fujise, K. Mizuno, Y. Ueda, S. Osada, S. Hirai, A. Takayanagi, N. Shimizu, M.K. Owada, H. Nakajima, S. Ohno, J. Biol. Chem. 269 (1994) 31642. [78] A.B. Oestreicher, H. Zwiers, P. Schotman, W.H. Gispen, Brain Res. Bull. 6 (1981) 145. [79] R.F. Akers, A. Routtenberg, Brain Res. 334 (1985) 147. [80] L. Aigner, P. Caroni, J. Cell Biol. 123 (1993) 417. [81] L. Aigner, S. Arber, J.P. Kapfhammer, T. Laux, C. Schneider, F. Botteri, H.R. Brenner, P. Caroni, Cell 83 (1995) 269. [82] S.A. Oehrlein, P.J. Parker, T. Herget, Biochem. J. 317 (1996) 219. [83] J.H. Hartwig, M. Thelen, A. Rosen, P.A. Janmey, A.C. Nairn, A. Aderem, Nature 356 (1992) 618. [84] M. Glaser, S. Wanaski, C.A. Buser, V. Boguslavsky, W. Rashidzada, A. Morris, M. Rebecchi, S.F. Scarlata, L.W. Runnels, G.D. Prestwich, J. Chen, A. Aderem, J. Ahn, S. McLaughlin, J. Biol. Chem. 271 (1996) 26187.
284
C. Larsson / Cellular Signalling 18 (2006) 276 – 284
[85] J. Wang, A. Arbuzova, G. Hangyas-Mihalyne, S. McLaughlin, J. Biol. Chem. 276 (2001) 5012. [86] T. Laux, K. Fukami, M. Thelen, T. Golub, D. Frey, P. Caroni, J. Cell Biol. 149 (2000) 1455. [87] Q. He, E.W. Dent, K.F. Meiri, J. Neurosci. 17 (1997) 3515. [88] J. Li, Z. Zhu, Z. Bao, J. Biol. Chem. 271 (1996) 12985. [89] M.M. Myat, S. Anderson, L.A. Allen, A. Aderem, Curr. Biol. 7 (1997) 611. [90] M.-H. Disatnik, S.C. Boutet, W. Pacio, A.Y. Chan, L.B. Ross, C.H. Lee, T.A. Rando, J. Cell Sci. 117 (2004) 4469. [91] X. Zhou, J. Li, J. Biol. Chem. 275 (2000) 20217. [92] Y. Matsuoka, X. Li, V. Bennett, Cell. Mol. Life Sci. 57 (2000) 884. [93] E. Ling, K. Gardner, V. Bennett, J. Biol. Chem. 261 (1986) 13875. [94] V. Bennett, K. Gardner, J.P. Steiner, J. Biol. Chem. 263 (1988) 5860. [95] A. Waseem, H.C. Palfrey, J. Cell Sci. 96 (1990) 93. [96] Y. Matsuoka, X. Li, V. Bennett, J. Cell Biol. 142 (1998) 485. [97] K.L. Barkalow, J.E. Italiano Jr., D.E. Chou, Y. Matsuoka, V. Bennett, J.H. Hartwig, J. Cell Biol. 161 (2003) 557. [98] J.C. Adams, Curr. Opin. Cell Biol. 16 (2004) 590. [99] J.C. Adams, J.D. Clelland, G.D.M. Collett, F. Matsumura, S. Yamashiro, L. Zhang, Mol. Biol. Cell 10 (1999) 4177. [100] N. Anilkumar, M. Parsons, R. Monk, T. Ng, J.C. Adams, EMBO J. 22 (2003) 5390. [101] A. Bretscher, K. Edwards, R.G. Fehon, Nat. Rev. Mol. Cell Biol. 3 (2002) 586.
[102] S.F. Pietromonaco, P.C. Simons, A. Altman, L. Elias, J. Biol. Chem. 273 (1998) 7594. [103] P.C. Simons, S.F. Pietromonaco, D. Reczek, A. Bretscher, L. Elias, Biochem. Biophys. Res. Commun. 253 (1998) 561. [104] J.W. Legg, C.A. Lewis, M. Parsons, T. Ng, C.M. Isacke, Nat. Cell Biol. 4 (2002) 399. [105] Y. Qian, J.M. Baisden, L. Cherezova, J.M. Summy, A. GuapponeKoay, X. Shi, T. Mast, J. Pustula, H.G. Zot, N. Mazloum, M.Y. Lee, D.C. Flynn, Mol. Biol. Cell 13 (2002) 2311. [106] A. Gatesman, V.G. Walker, J.M. Baisden, S.A. Weed, D.C. Flynn, Mol. Cell. Biol. 24 (2004) 7578. [107] D.K. Werth, J.E. Niedel, I. Pastan, J. Biol. Chem. 258 (1983) 11423. [108] W.H. Ziegler, U. Tigges, A. Zieseniss, B.M. Jockusch, J. Biol. Chem. 277 (2002) 7396. [109] M. Perez-Moreno, A. Avila, S. Islas, S. Sanchez, L. GonzalezMariscal, J. Cell Sci. 111 (1998) 3563. [110] R. Massoumi, A. Sjo¨lander, J. Cell Sci. 114 (2001) 1925. [111] A. Woods, J.R. Couchman, J. Cell Sci. 101 (1992) 277. [112] J. Li, K.L. O’Connor, G.H. Greeley Jr., P.J. Blackshear, C.M. Townsend Jr., B.M. Evers, J. Biol. Chem. 280 (2005) 8351. [113] S. Barry, D. Critchley, J. Cell Sci. 107 (1994) 2033. [114] M. Holinstat, D. Mehta, T. Kozasa, R.D. Minshall, A.B. Malik, J. Biol. Chem. 278 (2003) 28793. [115] P.A. Janmey, U. Lindberg, Nat. Rev. Mol. Cell. Biol. 5 (2004) 658.