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Recent advances in the protein kinase B signaling pathway
Recent advances in the protein kinase B signaling pathway James R Woodgett The phosphoinositide 30 kinase signaling pathway is activated in response to a plethora of growth factors and cytokines, and initiates a cascade of signaling events primarily via the induction of specific protein-serine/threonine kinases. Interest in the pathway has been driven by its frequent aberrant activation in disease and its impact on cell fate decisions owing to roles in survival signaling and metabolic control. There have been recent advances in our understanding of the primary components of this pathway, namely phosphoinositidedependent kinase-1, protein kinase B and glycogen synthase kinase-3, including insights into their mechanisms of regulation, substrate proteins and cellular functions. Addresses Ontario Cancer Institute/Princess Margaret Hospital, 610 University Avenue, Toronto, Ontario M5G 2 M9, Canada Corresponding author: Woodgett, James R ([email protected])
Current Opinion in Cell Biology 2005, 17:150–157 This review comes from a themed issue on Cell regulation Edited by Brian Hemmings and Peter Parker
0955-0674/$ – see front matter # 2005 Elsevier Ltd. All rights reserved. DOI 10.1016/j.ceb.2005.02.010
Introduction The phosphatidylinositol 30 kinase (PI3K) pathway is commonly activated in response to hormones and growth factors that act through cell surface receptors (see review by Dikic and co-workers in this issue). The pathway is also frequently activated in cancers via coupling to amplified or mutationally activated receptor-tyrosine kinases, amplification of genes encoding PI3K itself or downstream components, or loss of the phosphatidylinositol 30 lipid phosphatase (PTEN) [1–3]. For example, PTEN mutations occur in >75% of glioblastomas, and amplifications of PI3K catalytic domains have been found in ovarian and other cancers. Inhibition of PI3K signaling in such cancers leads to selective cell death, providing a potentially useful therapeutic intervention. The generation of 30 phosphorylated phosphoinositides by PI3K is restricted to membranes and thus this enzyme acts locally. Typically, proteins with specific phosphatidylinositol 30 phosphate (PIP3) binding motifs, such as Current Opinion in Cell Biology 2005, 17:150–157
certain pleckstrin homology (PH) or FYVE (using the single letter amino acid code) domains, are translocated to the source of PIP3 generation at the plasma or endosomal membranes, respectively, where they interact with other proteins or are, themselves, activated by PIP3 binding. Among the proteins to have PH domains with PIP3 specificity are two protein-serine kinases, phosphoinositide-dependant kinase 1 (PDK1) and protein kinase B (PKB; also known as Akt). PDK1 is crucial for the activation of PKB, as well as a series of other members of the kinase superfamily, which consists of cyclic AMP, GMP and protein kinase C (PKC) families (named the AGC family) (see below). PKBa was initially identified as a weak oncogene [4,5] and undergoes a complex mechanism of activation involving phosphorylation and conformational changes. Among the protein substrates of PKB are several pro-apoptotic proteins and another protein serine kinase termed glycogen synthase kinase-3 (GSK-3), which was also the first bona fide target of PKB to be identified [6]. GSK-3 targets many transcription factors, structural proteins and metabolic enzymes and has been implicated in cell fate determination and various human disorders including neurodegeneration, diabetes and cancer [7]. This review summarizes recent advances in our understanding of the regulation and functions of the PDK1/PKB/GSK-3 cascade, as well as highlighting the many significant gaps in our knowledge.
Mechanism protein kinase B activation Mammals harbour three isoforms of PKB, a, b and g (also known as Akt1, 2 and 3, respectively), which share a high degree of amino acid identity. These are composed of three functional domains: an N-terminal PH domain, a kinase domain and a C-terminal hydrophobic motif (HM) [8]. The catalytic domain of PKB is structurally similar to other protein kinases of the AGC family, including PKA and p70 ribosomal S6 kinase (p70 S6K), but is most similar to serum and glucocorticoid-regulated kinases (SGKs). Of note, SGK3 contains a related kinase domain and a hydrophobic motif but instead of a PH domain, it has a phox homology (PX) domain that exhibits a preference for phosphatidylinositides that are only phosphorylated at the 30 position such as phosphatidylinositol 3-phosphate. These lipids are predominantly found in the endosomal compartments of cells, with SGK3 being similarly distributed [9]. The importance of the PH domain to PKB activation was illustrated by the isolation of a point mutant of the only Drosophila PKB homologue, Dakt1. This mutant rescued the lethality caused by the deregulation of PIP3 that was induced by loss of the PTEN PIP3 phosphatase [10]. www.sciencedirect.com
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Figure 1
Plasma membrane PIP3 PH
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Activation process for PKB. In step 1, the generation of PIP3 at membrane sites of activated PI3K recruits both PDK1 and PKB into proximity. In step 2, phosphorylation of the HM (at Ser473) of PKB by PDK2 causes a conformational change in the kinase domain (KD) and also helps to align PDK1, which subsequently phosphorylates the T-loop of PKB (at Thr308) (step 3). Bi-phosphorylated PKB molecules (at the HM and T-loop sites) are fully active (step 4). Although phosphorylation of the HM of PKB is not requisite for PDK1 phosphorylation [13], it is likely to promote this event as well as encourage an active conformation state in the kinase domain.
A second important regulatory domain of PKB is the HM that is found in many other AGC kinases. The HM acts as a docking site for kinases such as PKC, p70 S6K, SGK and RSK to bind to PDK1 [11–13]. Mutation of the HM or use of competing peptides that bind to the HM attenuates phosphorylation of a residue in the activation loop (Tloop) of AGC kinases that is requisite for full activity. The HM also serves as an allosteric modulator of catalytic activity [12,14,15] by stabilizing a conformation of the catalytic domain by binding to hydrophobic and phosphate-binding pockets created by a cleft formed at the junction of the aB-helix, aC-helix and b5-sheet within the N-terminal lobe of the kinase domain. This structure has been termed the ‘PKC related kinase 2 (PRK2)interacting factor (PIF)-pocket’ by virtue of the initial identification of a homologous region on PDK1 that acts as the binding site for the HM of another AGC kinase, PRK2 [16]. Binding of the HM to this pocket increases kinase activity of PKB by tenfold. Of note, PDK1 has a PIF pocket but no HM. The crystal structure of the kinase domain of PKB has been solved, although this required deletion of the PH domain and substitution of the HM of PKB with the HM of PRK2 [15]. The HM of PRK2 contains a glutamic acid at the position of a serine in the HM of PKB. This serine (residue number 473 of PKBa) is phosphorylated in a PI3K-dependent manner and, like the T-loop residue (Thr308 in PKBa), is important for activation of PKB (Figure 1). For this reason, www.sciencedirect.com
phospho-specific antibodies that are selective for phosphorylated Thr308 or Ser473 have become widely used tools for assessing the activation states of PKB in tissues. However, the molecular mechanisms that lead to the phosphorylation of these sites and their role in PKB activation have been the source of significant controversy.
Protein kinase B kinases The identity of the protein kinase that targets the T-loop site of PKB is as well-established as PDK1. For example, embryonic stem cells that lack PDK1 or that express knock-in mutants within the PH or PIF pocket domains lose insulin-like growth factor 1-mediated phosphorylation of Thr308 but retain phosphorylation of Ser473 [17]. Mice that lack PDK1 die at embryonic day (E) 9.5, suffering from multiple abnormalities [18], but conditional knockout of the kinase in heart muscle has allowed examination of the role of this kinase in this tissue. Loss of PDK1 in the heart leads to heart failure within a few weeks of birth and myocytes from the conditional-knockout mice are unable to activate PKB or Rsk [19]. Similarly, genetic analysis in Drosophila has shown that a single PDK1 gene regulates PKB, Rsk and p70 S6K [20]. PDK1 expresses constitutive activity in cells [21]. This allows phosphorylation of several of its substrates in a non-regulated manner. Indeed, PDK1 is required for the activation of an array of AGC family kinases including Current Opinion in Cell Biology 2005, 17:150–157
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PKC isoforms, p70 S6K, Rsk and SGKs [17]. However, the C-terminal PH domain of PDK1 mediates translocation to sites of PIP3 generation following PI3K activation; the targets it phosphorylates at this location are therefore PI3K signal-dependent. PDK1 only encounters some of its kinase substrates, such as PKB, when at the membrane [22]. The binding of the PKB PH domain to PIP3 probably results in a conformational change that promotes the phosphorylation of the HM at Ser473. Phosphorylation of the HM of some AGC kinases is requisite for PDK1 to phosphorylate their T-loop sites as it causes an increase in the affinity for the PIF pocket on PDK1, thus helping to dock PDK1 in an orientation that promotes T-loop phosphorylation. This dependence might not be the case for PKB as PDK1 null cells expressing a mutant of the kinase with a defective PIF pocket that cannot interact with the HM can still phosphorylate PKB as long as it has an intact PH domain [13,23]. In addition, the affinity of the HM of PKB for the PIF pocket of PDK1 is lower than that for other PDK1 targets [24]. Thus, the juxtaposition of the PDK1 and PKB on the membrane might partially substitute for the HM docking mechanism. However, if this is the case, why does PKB retain a HM, why is it phosphorylated in a PI3K-dependent manner and why does mutation of Ser473 greatly reduce PKB activity? The answers are still unclear but probably relate to HM phosphorylation playing two roles in PKB activation. One is to accelerate the interaction with PDK1 and the subsequent T-loop phosphorylation (Figure 1). The PIF mutant knock-in experiments suggest that this role is not requisite, at least in embryonic stem cells [21], but do not exclude it as an important aspect of the activation process. The second role is in promoting a conformation of the PKB kinase domain that makes the enzyme maximally active. This is analogous to the role of the T-loop phosphorylation, which facilitates access to the ATP binding cleft, and structural studies have given evidence for such a conformational role. Analysis of AGC kinase homologues in budding yeast, Saccharomyces cerevisiae, revealed that for Ypk1, Ypk2, Pkc1 and Sch9, HM site phosphorylation only played a subtle role in activation and function [25]. Sch9 is most similar to PKB (albeit lacking a PH domain) and it remains to be determined whether these findings in yeast also apply to regulation of the mammalian PKBs.
Will the real phosphoinositol-dependent kinase-2 please stand up? The importance of HM phosphorylation would be easier to evaluate if the kinase(s) that phosphorylate this domain were known, as the effects of their inhibition on PKB activity and on T-loop phosphorylation could be assessed. There are several candidates for this long sought ‘PDK2’ activity but their relative physiological significance has been the source of much controversy [26]. Several protein kinases have been shown to be capable of phosphorylating Ser473 in vitro, but the use of inhibitors and cells that Current Opinion in Cell Biology 2005, 17:150–157
lack these kinases has raised questions as to whether they act as ‘PDK2’ in cells. Indeed, there are some data that support the idea that phosphorylation of the HM occurs by way of autophosphorylation [26]. However, in PDK1null cells Ser473 can still be induced, and because a lack of phosphorylation of Thr308 severely restricts PKB catalytic function it is not easy to reconcile this result with autophosphorylation, and it also excludes PDK1 [17,27]. There is also good evidence for phosphorylation of the HM of other AGC kinases by third party kinases. The best example is mammalian target of rapamycin (mTOR) phosphorylation of the HM of p70 S6K, which is crucial for full activation of this enzyme. The most compelling candidates for PDK2 include integrin-linked kinase (ILK), PKCbII and DNA-dependent protein kinase (DNA-PK), all of which have several confounding anomalies. ILK was first identified by its association with the intracellular domain of specific integrins [28]. Given the crucial role of extracellular matrix ligation in intracellular signaling, ILK is a good candidate for the coordination of matrix-based signals with growth factors. ILK has an unusual structure for a protein kinase and lacks several signature kinase residues. However, there are other atypical kinases that lack these conserved residues, and ILK has been purified as a myosin and associated protein kinase using conventional biochemistry [29]. Conditional knockout of ILK and RNAi suppression of the mammalian gene significantly reduces the level of PKB Ser473 phosphorylation [30]. Confounding these compelling data is the finding that loss of ILK function in Drosophila has a similar phenotype to loss of integrin function but has no effect on signaling [31]. Furthermore, mutations in the fly ILK that block kinase functions in the mammalian protein have no phenotypic consequences in Drosophila. Partial purification of a PDK2 fraction from membranes has also been shown to lack ILK protein [32]. ILK also binds several other proteins and has been proposed to act as an adaptor to couple these proteins to the sites of integrin adhesion. ILK might therefore play an important coordinating role for the actions of integrin, which probably include PKB regulation as a redundant function. In mast cells activated by phorbol myristate acetate or by the cross-linking of high affinity IgE receptors, PKCbII has been proposed to be the enzyme that mediates PKB phosphorylation at Ser473 [33]. Other isoforms of conventional PKCs did not share this property, nor was stem cell factor or interleukin 3-induced PKB phosphorylation affected in mast cells lacking PKCbII. Although PKCbII was capable of phosphorylating Ser473 of PKB in vitro, several other kinases share this capability so it is unclear whether stimulus-specific PDK2 function of this PKC is direct or indirect. Rather than take a candidate gene approach, several groups have attempted to characterize PDK2 activity www.sciencedirect.com
Protein kinase B signaling pathway Woodgett 153
within cell extracts [34,35]. Immunodepletion of PDK1 from a plasma membrane and cytosol extract blocked Tloop phosphorylation of PKB but had no effect on HM phosphorylation; this activity was found to be PI3Kdependent [34]. Assessment of the inhibitor sensitivity of HM phosphorylation revealed it to be insensitive to staurosporine, which potently interferes with PDK1 [32]. HM motif kinase activity has also been found to be associated with membrane raft fractions of cells that contain ILK. However, ILK-immunoprecipitates from these fractions did not phosphorylate Ser473 of PKB [32]. Purification of an activity capable of phosphorylating a peptide containing the PKB HM yielded a preparation containing the catalytic subunit of DNA-dependent kinase (DNA-PKCS) [36]. DNA-PK is an unusual kinase with a mass of 350 kDa that, like mTOR, is a member of the PI3K-like kinase family. Small interfering RNA knockdown of DNA-PK expression was found to reduce agonist-induced Ser473 phosphorylation; glioblastoma cells lacking DNA-PK had impaired phosphorylation of the PKB HM that was restored by re-expression of DNA-PKCS. However, although the Drosophila genome appears to lack a DNA-PK gene, the fly PKB homologue is regulated by phosphorylation of its HM (Ser505) [10]. In addition, Scid mice, which are mutant for DNA-PK, and DNA-PK knockout mice are severely immunodeficient [37] but do not appear to have defects associated with reduction of PKB activity (e.g. do not phenocopy PKBa or PKBb knockout mice). Ataxia telangiectasia mutant (ATM), a second DNA-damage-associated kinase of the PI3K-like kinase family, has also been implicated in the phosphorylation of the HM of PKB [38], which might explain possible redundancies with DNA-PK; however, in each case, knockdown of the kinase essentially ablated Ser473 phosphorylation of PKB. Clearly, despite enormous effort, there remain substantial questions regarding the nature of PDK2. From current data, it appears that its function is likely to be partially redundant, possibly with context- or agonist-dependent specificity. It is also intriguing that DNA-PK and ATM share sequence similarity to mTOR, which is an HM kinase for p70 S6K. However, unlike p70 S6K HM phosphorylation, phosphorylation of the Ser473 of PKB is insensitive to rapamycin. In yeast, the mTOR orthologues exists in two complexes, one of which is rapamycin-sensitive and contains a protein termed TORC1, which is homologous to Raptor in mammals [39]. The second complex is rapamycin-insensitive and contains a protein termed TORC2, which is similar to Rictor or mAVO3 (mammalian adheres voraciously 3) in mammals. This latter complex has recently been shown to mediate rapamycin-insensitive functions of mTOR on the actin cytoskeleton [40]. It remains to be determined whether or not this novel complex plays a role in regulating the HM of PKB. www.sciencedirect.com
Phosphoinositol-dependent kinase-1 regulation As mentioned above, PDK1 phosphorylates a variety of other protein kinases within the AGC subfamily. The catalytic activity of PDK1 is not acutely regulated and some targets are constitutively phosphorylated. However, the subcellular localization of PDK1 is signal-dependent. In addition to membrane targeting in response to PI3Kactivation mediated by its PH domain, PDK1 can also translocate to the nucleus in response to mitogens. Treatment of cells with leptomycin B, which inhibits the nuclear export receptor CRM1 (chromosomal region maintenance 1), causes accumulation of PDK1 in the nucleus [41]. PDK1 is constitutively phosphorylated on at least five residues, but PI3K stimulation leads to increased phosphorylation of a region proximal to the nuclear export sequence of the kinase; this phosphorylation is necessary for nuclear localization [42]. These findings support the idea that PDK1 can be selectively targeted to specific subcellular compartments in a signaldependent manner.
The importance of understanding protein kinase B regulation As mentioned above, PKB is commonly activated in tumors by a variety of mechanisms including inactivation of PTEN, amplification of receptors that couple to PI3K, or amplification of the signaling proteins themselves [43]. Among its substrates are proteins that promote apoptosis, such as Bad and the Foxo transcription factors. Phosphorylation of Bad induces association with a phosphoserine/ threonine-specific binding protein termed 14-3-3 and causes its dissociation from, and therefore the activation of, Bcl-2. Phosphorylation of Foxo promotes nuclear export, which prevents transcriptional induction of growth arrest and pro-apoptotic genes. Thus, PKB activation acts as a survival signal. However, activation of PKB is often insufficient for tumorigenesis. Although initially identified as a weak oncogene, expression of PKB in most cells does not lead to transformation. Expression of membrane-targeted, myristylated PKB alleles can lead to oncogenic transformation [44,45], but this form of the kinase is activated well beyond the levels observed in tumors. In several cases, transgenic expression of activated alleles of PKB reduces the apoptotic potential of cells and promotes growth without inducing transformation; this raises the question of why this kinase is commonly activated in tumors. Transgenic expression of polyoma middle T antigen (PyMT) in mammary epithelial cells leads to aggressive tumor formation within 50–60 days. By its association with Src-family kinases, PyMT becomes tyrosine phosphorylated and recruits SHC and PI3K subunits by way of their phosphotyrosine binding domains. SHC couples to the activation of mouse Son of sevenless (mSOS) and Ras, whereas PI3K induces PKB and mTOR activation. MutaCurrent Opinion in Cell Biology 2005, 17:150–157
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tions of PyMT that selectively interfere with the recruitment of PI3K result in a significant delay in tumor formation that was also qualitatively distinct, being hyperplastic, low grade and riddled with apoptotic cells. Mammary-gland-specific, transgenic expression of an activated allele of PKB (in which the two activatory phosphorylation sites were replaced by aspartic acid, mimicking phosphorylation) has only a minor phenotype on its own in which involution of the gland following weaning is delayed by a few days. However, in mice expressing the mutant PyMT that is uncoupled from PI3K activation, co-expression of the activated PKB allele fully restores aggressive tumorigenesis [46]. Thus, PKB activation cooperates with and supports Ras activation, which by itself induces self-limiting apoptosis as well as growth. The human epidermal growth factor receptorlike 2 (HER2; also known as neu) receptor tyrosine kinase also activates the PI3K and Ras pathways and is directly implicated in breast cancer. Interestingly, expression of activated PKB decreases the latency of HER2/neu tumors [47]. Synergy between the activation of Ras and PKB might be manifested at the level of recruitment of mRNAs into polysomes, leading to efficient translation of mRNAs encoding growth-promoting proteins [48]. PKB activation is therefore likely to play a supportive role in tumorigenesis. By raising the threshold of pro-apoptotic signaling required for induction of apoptotic pathways and by increasing the metabolic capacity of cells, this kinase helps to suppress the normally suicide-inducing effects of DNA lesions and genomic instability, which typically occur during the transformation process. In essence, activation of PKB helps provide the ‘cancer cat’ with nine lives! Normal cells do not need this protection as their genomes are stable and they are receiving positive signals from the matrix. Only tumor cells ‘become addicted’ to chronic PKB activity. For this reason, this kinase should be a useful therapeutic target. One caveat might apply to the effect that activation of PKBa has on promoting differentiation. In the models of breast cancer that is driven by PyMT and HER2/neu, expression of PKB increased the rate of tumor growth but severely reduced the metastatic potential of the tumors. Tumors with reduced metastasis were found to be more highly differentiated [47]. This might have unwanted consequences for general PKB inhibitors as reduction of the kinase activity might increase the metastatic potential of otherwise localized lesions. In contrast to the reduction in metastasis observed by PKBa expression, overexpression of PKBb is associated with increased invasion and spread [49]. Thus, isoform-selective PKB inhibitors might prove to be very useful [50].
Glycogen synthase kinase-3 GSK-3 was the first physiological target of PKB to be identified and although other substrates might be more closely related to the role of PKB in oncogenesis, GSK-3 Current Opinion in Cell Biology 2005, 17:150–157
is a key player in PKB signaling [6]. There are two isoforms of GSK-3, termed a and b, which are encoded by distinct genes. As for PKB, there is some evidence for distinct, but overlapping, roles and each isoform is similarly regulated. PKB phosphorylates a serine residue within the N-terminal domain (Ser21 in GSK-3a and Ser9 in GSK-3b). This inhibits the kinase. The two isoforms are ubiquitously expressed and are highly conserved throughout eukaryotes. Indeed, clues to the physiological functions of this kinase have often derived from genetic analysis of simpler organisms. Unlike PKB, GSK3 is largely activated in resting, unstimulated cells. Its substrates are therefore highly phosphorylated in the absence of agonists. GSK-3 phosphorylates a wide array of proteins including metabolic enzymes, structural proteins and transcription factors. Where studied, the effect of GSK-3 phosphorylation tends to inactivate the substrate protein. Therefore, PKB-dependent inactivation of GSK-3 promotes the dephosphorylation and the activation of the many substrates of GSK-3 [7].
Priming site phosphorylation Many GSK-3 substrates share a peculiarity in that their phosphorylation by GSK-3 requires prior phosphorylation at a proximal residue by another protein kinase. The molecular basis of this unusual prerequisite for priming phosphorylation became apparent upon determination of the crystal structure of GSK-3b [51,52], as well as by some elegant mutagenesis [53]. Kinases related to GSK-3, such as cyclin-dependent kinases, p38 mitogen-activated protein kinases, and extracellular signal-dependent protein kinases, require phosphorylated residues on their T-loops loops for activity. A phosphothreonine and/or a phosphotyrosine aligns key b-strand and a-helical structures in the two lobes to allow entry of substrates to their catalytic sites. The T-loop of GSK-3 is phosphorylated at a single tyrosine (Tyr216 and Tyr279 for GSK-3b and GSK-3a, respectively). However, this site is highly phosphorylated (probably by an autophosphorylation mechanism [54]). In the crystal structure of GSK-3b, the conformation of the T-loop tyrosine does not directly preclude access to the ATP binding site when unphosphorylated, and phosphorylation only moderately increases activity. However, the relative positions of the N and C-terminal lobes of the kinase domain remain in an inactive conformation until the binding of a pre-phosphorylated substrate. The priming phosphate of the substrate binds to a positively charged pocket comprising of Arg96 and Arg180, as well as Lys205 (GSK-3b residues). This aligns the catalytic lobes into an optimal conformation for activity as well as orienting the substrate phospho-acceptor residue (Figure 2). The primed phosphate-binding pocket on GSK-3 serves another important regulatory function. Phosphorylation of the N-terminal domain at Ser21 or at Ser9 by PKB facilitates intramolecular binding of this residue to the www.sciencedirect.com
Protein kinase B signaling pathway Woodgett 155
Figure 2
(a) GSK-3 GSK-3 P Primed substrate
Substrate
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(b) Primed phosphate docking pocket
PK(A, B or C) Ser9/21
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Role of the phosphate-binding pocket of GSK-3 in substrate recognition and regulation. In (a) proteins only become substrates for GSK-3 upon phosphorylation by a priming kinase. GSK-3 binds to the phosphorylated substrate by way of a phosphate-binding pocket that causes an activating conformational change and properly aligns the substrate for phosphorylation. The phosphate introduced by GSK-3 can act as a priming site for subsequent phosphorylations in some targets (such as glycogen synthase and b-catenin). In (b) the phosphate-docking site can also bind to the intramolecular phosphate located at Ser9 (GSK-3b) or Ser21 (GSK-3a) that is introduced by other protein kinase (such as PKB) located within the N-terminal of GSK-3. This results in intramolecular occupation of the docking site by the N-terminal domain, precluding intermolecular binding to the primed phosphates of substrate proteins, effectively shutting down the enzyme.
substrate priming phosphate-binding pocket and by doing so, excludes binding of substrates, to effectively inhibit GSK-3 activity. In addition to PKB, several other protein kinases phosphorylate the inhibitory N-terminal site of GSK-3, including cyclic AMP-dependent kinase and certain PKCs (Figure 2) [55,56]. The priming phosphorylation site on substrates has a potential regulatory function as signals that modulate the degree of phosphorylation of these residues will affect their binding to GSK3. However, most priming sites are constitutively phosphorylated and are not signal dependent. In yeast and Dictyostelium, the substrates of the GSK-3 orthologues require priming but the kinases themselves do not harbour N-terminal regulatory phosphorylation sites, suggesting this level of regulation was a more recent event than substrate priming.
phosphate-binding pocket (PIF pocket) on PDK1 aids in its processing of the T-loop phosphorylation of PKB. The HM of PKB then assists in stabilizing an active conformation by intramolecular binding to the catalytic domain. A phosphate-binding pocket on GSK-3 directs it to specific substrates as well as providing a means of regulation through the intramolecular binding of a regulatory domain. Protein phosphorylation acts in this cascade as a selective binding mechanism that is analogous to Srchomology-2 (SH2) and phosphotyrosine binding (PTB)domain binding to phosphotyrosine at the level of receptor tyrosine kinases and their adapters. The remarkable degree of complexity in the regulation of this cascade, and of PKB in particular, is likely to reflect the dramatic consequences of its inappropriate activation as well as its core role in the engineered response to a variety of mitogens and growth factors.
Conclusions Analysis of the PDK/PKB/GSK-3 cascade reveals several recurring themes. Although these proteins couple to multiple pathways, their specific interactions are mediated through selective phosphate binding sites and recruitment to specific intracellular domains. The www.sciencedirect.com
Acknowledgements The author is supported by grants from the Canadian Institutes of Health Research and the Canadian Cancer Society, and is a Howard Hughes Medical Institute International Scholar. Current Opinion in Cell Biology 2005, 17:150–157
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PDK1 in cardiac muscle results in heart failure and increased sensitivity to hypoxia. EMBO J 2003, 22:4666-4676. 20. Rintelen F, Stocker H, Thomas G, Hafen E: PDK1 regulates growth through Akt and S6K in Drosophila. Proc Natl Acad Sci USA 2001, 98:15020-15025. 21. Mora A, Komander D, van Aalten DM, Alessi DR: PDK1, the master regulator of AGC kinase signal transduction. Semin Cell Dev Biol 2004, 15:161-170. 22. Scheid MP, Marignani PA, Woodgett JR: Multiple phosphoinositide 3-kinase-dependent steps in activation of protein kinase B. Mol Cell Biol 2002, 22:6247-6260. 23. Collins BJ, Deak M, Arthur JS, Armit LJ, Alessi DR: In vivo role of the PIF-binding docking site of PDK1 defined by knock-in mutation. EMBO J 2003, 22:4202-4211. This paper demonstrates, in an elegant manner, the importance of the PIF-pocket of PDK1 on phosphorylation of certain of its substrate proteins in embryonic stem cells. 24. Frodin M, Antal TL, Dummler BA, Jensen CJ, Deak M, Gammeltoft S, Biondi RM: A phosphoserine/threonine-binding pocket in AGC kinases and PDK1 mediates activation by hydrophobic motif phosphorylation. EMBO J 2002, 21:5396-5407. 25. Roelants FM, Torrance PD, Thorner J: Differential roles of PDK1and PDK2-phosphorylation sites in the yeast AGC kinases Ypk1, Pkc1 and Sch9. Microbiology 2004, 150:3289-3304. 26. Chan TO, Tsichlis PN: PDK2: a complex tail in one Akt. Science STKE. 2001, PE1. 27. Hill MM, Andjelkovic M, Brazil DP, Ferrari S, Fabbro D, Hemmings BA: Insulin-stimulated protein kinase B phosphorylation on Ser-473 is independent of its activity and occurs through a staurosporine-insensitive kinase. J Biol Chem 2001, 276:25643-25646. 28. Persad S, Dedhar S: The role of integrin-linked kinase (ILK) in cancer progression. Cancer Metastasis Rev 2003, 22:375-384. 29. Deng JT, Sutherland C, Brautigan DL, Eto M, Walsh MP: Phosphorylation of the myosin phosphatase inhibitors, CPI-17 and PHI-1, by integrin-linked kinase. Biochem J 2002, 367:517-524. 30. Troussard AA, Mawji NM, Ong C, Mui A, St-Arnaud R, Dedhar S: Conditional knock-out of integrin-linked kinase demonstrates an essential role in protein kinase B/Akt activation. J Biol Chem 2003, 278:22374-22378. This study provides genetic evidence for a role of ILK kinase activity in the phosphorylation of PKB at Ser473, using conditional knockout and siRNA approaches. 31. Zervas CG, Gregory SL, Brown NH: Drosophila integrin-linked kinase is required at sites of integrin adhesion to link the cytoskeleton to the plasma membrane. J Cell Biol 2001, 152:1007-1018. 32. Hill MM, Feng J, Hemmings BA: Identification of a plasma membrane Raft-associated PKB Ser473 kinase activity that is distinct from ILK and PDK1. Curr Biol 2002, 12:1251-1255. 33. Kawakami Y, Nishimoto H, Kitaura J, Maeda-Yamamoto M, Kato RM, Littman DR, Rawlings DJ, Kawakami T: Protein kinase C bII regulates Akt phosphorylation on Ser-473 in a cell-type- and stimulus-specific fashion. J Biol Chem 2004, 279:47720-47725. 34. Hresko RC, Murata H, Mueckler M: Phosphoinositidedependent kinase-2 is a distinct protein kinase enriched in a novel cytoskeletal fraction associated with adipocyte plasma membranes. J Biol Chem 2003, 278:21615-21622.
17. Williams MR, Arthur JS, Balendran A, van der Kaay J, Poli V, Cohen P, Alessi DR: The role of 3-phosphoinositide-dependent protein kinase 1 in activating AGC kinases defined in embryonic stem cells. Curr Biol 2000, 10:439-448.
35. Hodgkinson CP, Sale EM, Sale GJ: Characterization of PDK2 activity against protein kinase Bg. Biochemistry 2002, 41:10351-10359.
18. Lawlor MA, Mora A, Ashby PR, Williams MR, Murray-Tait V, Malone L, Prescott AR, Lucocq JM, Alessi DR: Essential role of PDK1 in regulating cell size and development in mice. EMBO J 2002, 21:3728-3738.
36. Feng J, Park J, Cron P, Hess D, Hemmings BA: Identification of a PKB/Akt hydrophobic motif Ser-473 kinase as DNAdependent protein kinase. J Biol Chem 2004, 279:41189-41196. One of several important papers characterizing putative PDK2 activities.
19. Mora A, Davies AM, Bertrand L, Sharif I, Budas GR, Jovanovic S, Mouton V, Kahn CR, Lucocq JM, Gray GA et al.: Deficiency of
37. Gao Y, Chaudhuri J, Zhu C, Davidson L, Weaver DT, Alt FW: A targeted DNA-PKcs-null mutation reveals DNA-PK-
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Protein kinase B signaling pathway Woodgett 157
independent functions for KU in V(D)J recombination. Immunity 1998, 9:367-376. 38. Guinea Viniegra J, Martinez N, Modirassari P, Hernandez Losa J, Parada Cobo C, Sanchez-Arevalo Lobo VJ, Avceves-Luquero CI, Alavrez-Vallina L, Ramon Y Cajal S, Rojas JM, Sanchez-Prieto R: Full activation of PKB/Akt in response to insulin or ionizing radiation is mediated through ATM. J Biol Chem 2005, in press. 39. Loewith R, Jacinto E, Wullschleger S, Lorberg A, Crespo JL, Bonenfant D, Oppliger W, Jenoe P, Hall MN: Two TOR complexes, only one of which is rapamycin sensitive, have distinct roles in cell growth control. Mol Cell 2002, 10:457-468. 40. Jacinto E, Loewith R, Schmidt A, Lin S, Ruegg MA, Hall A, Hall MN: Mammalian TOR complex 2 controls the actin cytoskeleton and is rapamycin insensitive. Nat Cell Biol 2004, 6:1122-1128. This paper demonstrates the existence of a second mTOR complex in mammalian cells that is not inhibited by rapamycin, which raises the possibility of roles for mTOR in rapamycin-insensitive processes. 41. Lim MA, Kikani CK, Wick MJ, Dong LQ: Nuclear translocation of 3(-phosphoinositide-dependent protein kinase 1 (PDK-1): a potential regulatory mechanism for PDK-1 function. Proc Natl Acad Sci USA 2003, 100:14006-14011. This paper demonstrated that nuclear transport of PDK-1 has the potential to be regulated rather than being a passive process and identified a functional nuclear export sequence (NES) in the molecule. 42. Scheid MP, Parsons M, Woodgett JR: Phosphoinositide dependent phosphorylation of PDK1 regulates nuclear translocation. Mol Cell Biol 2005, in press. This paper identifies PI3K-dependent phosphorylation of PDK-1 to be a means to regulate its nuclear entry and exit, providing another layer of the regulation to this protein kinase. 43. Brazil DP, Yang ZZ, Hemmings BA: Advances in protein kinase B signalling: AKTion on multiple fronts. Trends Biochem Sci 2004, 29:233-242. 44. Mende I, Malstrom S, Tsichlis PN, Vogt PK, Aoki M: Oncogenic transformation induced by membrane-targeted Akt2 and Akt3. Oncogene 2001, 20:4419-4423. 45. Majumder PK, Yeh JJ, George DJ, Febbo PG, Kum J, Xue Q, Bikoff R, Ma H, Kantoff PW, Golub TR et al.: Prostate intraepithelial neoplasia induced by prostate restricted Akt activation: the MPAKT model. Proc Natl Acad Sci USA 2003, 100:7841-7846. 46. Hutchinson J, Jin J, Cardiff RD, Woodgett JR, Muller WJ: Activation of Akt (protein kinase B) in mammary epithelium provides a critical cell survival signal required for tumor progression. Mol Cell Biol 2001, 21:2203-2212. 47. Hutchinson JN, Jin J, Cardiff RD, Woodgett JR, Muller WJ: Activation of Akt-1 (PKB-a) can accelerate ErbB-2-mediated
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mammary tumorigenesis but suppresses tumor invasion. Cancer Res 2004, 64:3171-3178. 48. Rajasekhar VK, Viale A, Socci ND, Wiedmann M, Hu X, Holland EC: Oncogenic Ras and Akt signaling contribute to glioblastoma formation by differential recruitment of existing mRNAs to polysomes. Mol Cell 2003, 12:889-901. 49. Arboleda MJ, Lyons JF, Kabbinavar FF, Bray MR, Snow BE, Ayala R, Danino M, Karlan BY, Slamon DJ: Overexpression of AKT2/protein kinase Bb leads to up-regulation of b1 integrins, increased invasion, and metastasis of human breast and ovarian cancer cells. Cancer Res 2003, 63:196-206. This study provides insight into the biological consequences of increased levels of the b isoform of PKB on tumor cell properties. 50. Barnett SF, Defeo-Jones D, Fu S, Hancock PJ, Haskell KM, Jones RE, Kahana JA, Kral AM, Leander K, Lee LL et al.: Identification and characterization of pleckstrin homology domain dependent and isozyme specific Akt inhibitors. Biochem J 2005, 385:399-408. This paper from Merck introduces isoform-selective PKB inhibitors that act through binding to both the PH and protein kinase domains. These reagents should prove very useful in understanding PKB functions. 51. Dajani R, Fraser E, Roe SM, Young N, Good V, Dale TC, Pearl LH: Crystal structure of glycogen synthase kinase 3 b: structural basis for phosphate-primed substrate specificity and autoinhibition. Cell 2001, 105:721-732. 52. ter Haar E, Coll JT, Austen DA, Hsiao HM, Swenson L, Jain J: Structure of GSK3b reveals a primed phosphorylation mechanism. Nat Struct Biol 2001, 8:593-596. 53. Frame S, Cohen P, Biondi RM: A common phosphate binding site explains the unique substrate specificity of GSK3 and its inactivation by phosphorylation. Mol Cell 2001, 7:1321-1327. 54. Cole A, Frame S, Cohen P: Further evidence that the tyrosine phosphorylation of glycogen synthase kinase-3 (GSK3) in mammalian cells is an autophosphorylation event. Biochem J 2004, 377:249-255. 55. Fang X, Lu Y, Yu SX, Lu Y, Bast RC, Woodgett JR, Mills GB: Phosphorylation and inactivation of glycogen synthase kinase-3 by both protein kinase A and protein kinase B. Proc Natl Acad Sci USA 2000, 97:11960-11965. 56. Fang X, Yu S, Tanyi JL, Lu Y, Woodgett JR, Mills GB: Convergence of multiple signaling cascades at glycogen synthase kinase 3: Edg receptor-mediated phosphorylation and inactivation by lysophosphatidic acid through a protein kinase C-dependent intracellular pathway. Mol Cell Biol 2002, 22:2099-2110.
Current Opinion in Cell Biology 2005, 17:150–157
Current Opinion in Cell Biology 2005, 17:150–157 This review comes from a themed issue on Cell regulation Edited by Brian Hemmings and Peter Parker
0955-0674/$ – see front matter # 2005 Elsevier Ltd. All rights reserved. DOI 10.1016/j.ceb.2005.02.010
Introduction The phosphatidylinositol 30 kinase (PI3K) pathway is commonly activated in response to hormones and growth factors that act through cell surface receptors (see review by Dikic and co-workers in this issue). The pathway is also frequently activated in cancers via coupling to amplified or mutationally activated receptor-tyrosine kinases, amplification of genes encoding PI3K itself or downstream components, or loss of the phosphatidylinositol 30 lipid phosphatase (PTEN) [1–3]. For example, PTEN mutations occur in >75% of glioblastomas, and amplifications of PI3K catalytic domains have been found in ovarian and other cancers. Inhibition of PI3K signaling in such cancers leads to selective cell death, providing a potentially useful therapeutic intervention. The generation of 30 phosphorylated phosphoinositides by PI3K is restricted to membranes and thus this enzyme acts locally. Typically, proteins with specific phosphatidylinositol 30 phosphate (PIP3) binding motifs, such as Current Opinion in Cell Biology 2005, 17:150–157
certain pleckstrin homology (PH) or FYVE (using the single letter amino acid code) domains, are translocated to the source of PIP3 generation at the plasma or endosomal membranes, respectively, where they interact with other proteins or are, themselves, activated by PIP3 binding. Among the proteins to have PH domains with PIP3 specificity are two protein-serine kinases, phosphoinositide-dependant kinase 1 (PDK1) and protein kinase B (PKB; also known as Akt). PDK1 is crucial for the activation of PKB, as well as a series of other members of the kinase superfamily, which consists of cyclic AMP, GMP and protein kinase C (PKC) families (named the AGC family) (see below). PKBa was initially identified as a weak oncogene [4,5] and undergoes a complex mechanism of activation involving phosphorylation and conformational changes. Among the protein substrates of PKB are several pro-apoptotic proteins and another protein serine kinase termed glycogen synthase kinase-3 (GSK-3), which was also the first bona fide target of PKB to be identified [6]. GSK-3 targets many transcription factors, structural proteins and metabolic enzymes and has been implicated in cell fate determination and various human disorders including neurodegeneration, diabetes and cancer [7]. This review summarizes recent advances in our understanding of the regulation and functions of the PDK1/PKB/GSK-3 cascade, as well as highlighting the many significant gaps in our knowledge.
Mechanism protein kinase B activation Mammals harbour three isoforms of PKB, a, b and g (also known as Akt1, 2 and 3, respectively), which share a high degree of amino acid identity. These are composed of three functional domains: an N-terminal PH domain, a kinase domain and a C-terminal hydrophobic motif (HM) [8]. The catalytic domain of PKB is structurally similar to other protein kinases of the AGC family, including PKA and p70 ribosomal S6 kinase (p70 S6K), but is most similar to serum and glucocorticoid-regulated kinases (SGKs). Of note, SGK3 contains a related kinase domain and a hydrophobic motif but instead of a PH domain, it has a phox homology (PX) domain that exhibits a preference for phosphatidylinositides that are only phosphorylated at the 30 position such as phosphatidylinositol 3-phosphate. These lipids are predominantly found in the endosomal compartments of cells, with SGK3 being similarly distributed [9]. The importance of the PH domain to PKB activation was illustrated by the isolation of a point mutant of the only Drosophila PKB homologue, Dakt1. This mutant rescued the lethality caused by the deregulation of PIP3 that was induced by loss of the PTEN PIP3 phosphatase [10]. www.sciencedirect.com
Protein kinase B signaling pathway Woodgett 151
Figure 1
Plasma membrane PIP3 PH
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PH P HM KD P PKB (active) Current Opinion in Cell Biology
Activation process for PKB. In step 1, the generation of PIP3 at membrane sites of activated PI3K recruits both PDK1 and PKB into proximity. In step 2, phosphorylation of the HM (at Ser473) of PKB by PDK2 causes a conformational change in the kinase domain (KD) and also helps to align PDK1, which subsequently phosphorylates the T-loop of PKB (at Thr308) (step 3). Bi-phosphorylated PKB molecules (at the HM and T-loop sites) are fully active (step 4). Although phosphorylation of the HM of PKB is not requisite for PDK1 phosphorylation [13], it is likely to promote this event as well as encourage an active conformation state in the kinase domain.
A second important regulatory domain of PKB is the HM that is found in many other AGC kinases. The HM acts as a docking site for kinases such as PKC, p70 S6K, SGK and RSK to bind to PDK1 [11–13]. Mutation of the HM or use of competing peptides that bind to the HM attenuates phosphorylation of a residue in the activation loop (Tloop) of AGC kinases that is requisite for full activity. The HM also serves as an allosteric modulator of catalytic activity [12,14,15] by stabilizing a conformation of the catalytic domain by binding to hydrophobic and phosphate-binding pockets created by a cleft formed at the junction of the aB-helix, aC-helix and b5-sheet within the N-terminal lobe of the kinase domain. This structure has been termed the ‘PKC related kinase 2 (PRK2)interacting factor (PIF)-pocket’ by virtue of the initial identification of a homologous region on PDK1 that acts as the binding site for the HM of another AGC kinase, PRK2 [16]. Binding of the HM to this pocket increases kinase activity of PKB by tenfold. Of note, PDK1 has a PIF pocket but no HM. The crystal structure of the kinase domain of PKB has been solved, although this required deletion of the PH domain and substitution of the HM of PKB with the HM of PRK2 [15]. The HM of PRK2 contains a glutamic acid at the position of a serine in the HM of PKB. This serine (residue number 473 of PKBa) is phosphorylated in a PI3K-dependent manner and, like the T-loop residue (Thr308 in PKBa), is important for activation of PKB (Figure 1). For this reason, www.sciencedirect.com
phospho-specific antibodies that are selective for phosphorylated Thr308 or Ser473 have become widely used tools for assessing the activation states of PKB in tissues. However, the molecular mechanisms that lead to the phosphorylation of these sites and their role in PKB activation have been the source of significant controversy.
Protein kinase B kinases The identity of the protein kinase that targets the T-loop site of PKB is as well-established as PDK1. For example, embryonic stem cells that lack PDK1 or that express knock-in mutants within the PH or PIF pocket domains lose insulin-like growth factor 1-mediated phosphorylation of Thr308 but retain phosphorylation of Ser473 [17]. Mice that lack PDK1 die at embryonic day (E) 9.5, suffering from multiple abnormalities [18], but conditional knockout of the kinase in heart muscle has allowed examination of the role of this kinase in this tissue. Loss of PDK1 in the heart leads to heart failure within a few weeks of birth and myocytes from the conditional-knockout mice are unable to activate PKB or Rsk [19]. Similarly, genetic analysis in Drosophila has shown that a single PDK1 gene regulates PKB, Rsk and p70 S6K [20]. PDK1 expresses constitutive activity in cells [21]. This allows phosphorylation of several of its substrates in a non-regulated manner. Indeed, PDK1 is required for the activation of an array of AGC family kinases including Current Opinion in Cell Biology 2005, 17:150–157
152 Cell regulation
PKC isoforms, p70 S6K, Rsk and SGKs [17]. However, the C-terminal PH domain of PDK1 mediates translocation to sites of PIP3 generation following PI3K activation; the targets it phosphorylates at this location are therefore PI3K signal-dependent. PDK1 only encounters some of its kinase substrates, such as PKB, when at the membrane [22]. The binding of the PKB PH domain to PIP3 probably results in a conformational change that promotes the phosphorylation of the HM at Ser473. Phosphorylation of the HM of some AGC kinases is requisite for PDK1 to phosphorylate their T-loop sites as it causes an increase in the affinity for the PIF pocket on PDK1, thus helping to dock PDK1 in an orientation that promotes T-loop phosphorylation. This dependence might not be the case for PKB as PDK1 null cells expressing a mutant of the kinase with a defective PIF pocket that cannot interact with the HM can still phosphorylate PKB as long as it has an intact PH domain [13,23]. In addition, the affinity of the HM of PKB for the PIF pocket of PDK1 is lower than that for other PDK1 targets [24]. Thus, the juxtaposition of the PDK1 and PKB on the membrane might partially substitute for the HM docking mechanism. However, if this is the case, why does PKB retain a HM, why is it phosphorylated in a PI3K-dependent manner and why does mutation of Ser473 greatly reduce PKB activity? The answers are still unclear but probably relate to HM phosphorylation playing two roles in PKB activation. One is to accelerate the interaction with PDK1 and the subsequent T-loop phosphorylation (Figure 1). The PIF mutant knock-in experiments suggest that this role is not requisite, at least in embryonic stem cells [21], but do not exclude it as an important aspect of the activation process. The second role is in promoting a conformation of the PKB kinase domain that makes the enzyme maximally active. This is analogous to the role of the T-loop phosphorylation, which facilitates access to the ATP binding cleft, and structural studies have given evidence for such a conformational role. Analysis of AGC kinase homologues in budding yeast, Saccharomyces cerevisiae, revealed that for Ypk1, Ypk2, Pkc1 and Sch9, HM site phosphorylation only played a subtle role in activation and function [25]. Sch9 is most similar to PKB (albeit lacking a PH domain) and it remains to be determined whether these findings in yeast also apply to regulation of the mammalian PKBs.
Will the real phosphoinositol-dependent kinase-2 please stand up? The importance of HM phosphorylation would be easier to evaluate if the kinase(s) that phosphorylate this domain were known, as the effects of their inhibition on PKB activity and on T-loop phosphorylation could be assessed. There are several candidates for this long sought ‘PDK2’ activity but their relative physiological significance has been the source of much controversy [26]. Several protein kinases have been shown to be capable of phosphorylating Ser473 in vitro, but the use of inhibitors and cells that Current Opinion in Cell Biology 2005, 17:150–157
lack these kinases has raised questions as to whether they act as ‘PDK2’ in cells. Indeed, there are some data that support the idea that phosphorylation of the HM occurs by way of autophosphorylation [26]. However, in PDK1null cells Ser473 can still be induced, and because a lack of phosphorylation of Thr308 severely restricts PKB catalytic function it is not easy to reconcile this result with autophosphorylation, and it also excludes PDK1 [17,27]. There is also good evidence for phosphorylation of the HM of other AGC kinases by third party kinases. The best example is mammalian target of rapamycin (mTOR) phosphorylation of the HM of p70 S6K, which is crucial for full activation of this enzyme. The most compelling candidates for PDK2 include integrin-linked kinase (ILK), PKCbII and DNA-dependent protein kinase (DNA-PK), all of which have several confounding anomalies. ILK was first identified by its association with the intracellular domain of specific integrins [28]. Given the crucial role of extracellular matrix ligation in intracellular signaling, ILK is a good candidate for the coordination of matrix-based signals with growth factors. ILK has an unusual structure for a protein kinase and lacks several signature kinase residues. However, there are other atypical kinases that lack these conserved residues, and ILK has been purified as a myosin and associated protein kinase using conventional biochemistry [29]. Conditional knockout of ILK and RNAi suppression of the mammalian gene significantly reduces the level of PKB Ser473 phosphorylation [30]. Confounding these compelling data is the finding that loss of ILK function in Drosophila has a similar phenotype to loss of integrin function but has no effect on signaling [31]. Furthermore, mutations in the fly ILK that block kinase functions in the mammalian protein have no phenotypic consequences in Drosophila. Partial purification of a PDK2 fraction from membranes has also been shown to lack ILK protein [32]. ILK also binds several other proteins and has been proposed to act as an adaptor to couple these proteins to the sites of integrin adhesion. ILK might therefore play an important coordinating role for the actions of integrin, which probably include PKB regulation as a redundant function. In mast cells activated by phorbol myristate acetate or by the cross-linking of high affinity IgE receptors, PKCbII has been proposed to be the enzyme that mediates PKB phosphorylation at Ser473 [33]. Other isoforms of conventional PKCs did not share this property, nor was stem cell factor or interleukin 3-induced PKB phosphorylation affected in mast cells lacking PKCbII. Although PKCbII was capable of phosphorylating Ser473 of PKB in vitro, several other kinases share this capability so it is unclear whether stimulus-specific PDK2 function of this PKC is direct or indirect. Rather than take a candidate gene approach, several groups have attempted to characterize PDK2 activity www.sciencedirect.com
Protein kinase B signaling pathway Woodgett 153
within cell extracts [34,35]. Immunodepletion of PDK1 from a plasma membrane and cytosol extract blocked Tloop phosphorylation of PKB but had no effect on HM phosphorylation; this activity was found to be PI3Kdependent [34]. Assessment of the inhibitor sensitivity of HM phosphorylation revealed it to be insensitive to staurosporine, which potently interferes with PDK1 [32]. HM motif kinase activity has also been found to be associated with membrane raft fractions of cells that contain ILK. However, ILK-immunoprecipitates from these fractions did not phosphorylate Ser473 of PKB [32]. Purification of an activity capable of phosphorylating a peptide containing the PKB HM yielded a preparation containing the catalytic subunit of DNA-dependent kinase (DNA-PKCS) [36]. DNA-PK is an unusual kinase with a mass of 350 kDa that, like mTOR, is a member of the PI3K-like kinase family. Small interfering RNA knockdown of DNA-PK expression was found to reduce agonist-induced Ser473 phosphorylation; glioblastoma cells lacking DNA-PK had impaired phosphorylation of the PKB HM that was restored by re-expression of DNA-PKCS. However, although the Drosophila genome appears to lack a DNA-PK gene, the fly PKB homologue is regulated by phosphorylation of its HM (Ser505) [10]. In addition, Scid mice, which are mutant for DNA-PK, and DNA-PK knockout mice are severely immunodeficient [37] but do not appear to have defects associated with reduction of PKB activity (e.g. do not phenocopy PKBa or PKBb knockout mice). Ataxia telangiectasia mutant (ATM), a second DNA-damage-associated kinase of the PI3K-like kinase family, has also been implicated in the phosphorylation of the HM of PKB [38], which might explain possible redundancies with DNA-PK; however, in each case, knockdown of the kinase essentially ablated Ser473 phosphorylation of PKB. Clearly, despite enormous effort, there remain substantial questions regarding the nature of PDK2. From current data, it appears that its function is likely to be partially redundant, possibly with context- or agonist-dependent specificity. It is also intriguing that DNA-PK and ATM share sequence similarity to mTOR, which is an HM kinase for p70 S6K. However, unlike p70 S6K HM phosphorylation, phosphorylation of the Ser473 of PKB is insensitive to rapamycin. In yeast, the mTOR orthologues exists in two complexes, one of which is rapamycin-sensitive and contains a protein termed TORC1, which is homologous to Raptor in mammals [39]. The second complex is rapamycin-insensitive and contains a protein termed TORC2, which is similar to Rictor or mAVO3 (mammalian adheres voraciously 3) in mammals. This latter complex has recently been shown to mediate rapamycin-insensitive functions of mTOR on the actin cytoskeleton [40]. It remains to be determined whether or not this novel complex plays a role in regulating the HM of PKB. www.sciencedirect.com
Phosphoinositol-dependent kinase-1 regulation As mentioned above, PDK1 phosphorylates a variety of other protein kinases within the AGC subfamily. The catalytic activity of PDK1 is not acutely regulated and some targets are constitutively phosphorylated. However, the subcellular localization of PDK1 is signal-dependent. In addition to membrane targeting in response to PI3Kactivation mediated by its PH domain, PDK1 can also translocate to the nucleus in response to mitogens. Treatment of cells with leptomycin B, which inhibits the nuclear export receptor CRM1 (chromosomal region maintenance 1), causes accumulation of PDK1 in the nucleus [41]. PDK1 is constitutively phosphorylated on at least five residues, but PI3K stimulation leads to increased phosphorylation of a region proximal to the nuclear export sequence of the kinase; this phosphorylation is necessary for nuclear localization [42]. These findings support the idea that PDK1 can be selectively targeted to specific subcellular compartments in a signaldependent manner.
The importance of understanding protein kinase B regulation As mentioned above, PKB is commonly activated in tumors by a variety of mechanisms including inactivation of PTEN, amplification of receptors that couple to PI3K, or amplification of the signaling proteins themselves [43]. Among its substrates are proteins that promote apoptosis, such as Bad and the Foxo transcription factors. Phosphorylation of Bad induces association with a phosphoserine/ threonine-specific binding protein termed 14-3-3 and causes its dissociation from, and therefore the activation of, Bcl-2. Phosphorylation of Foxo promotes nuclear export, which prevents transcriptional induction of growth arrest and pro-apoptotic genes. Thus, PKB activation acts as a survival signal. However, activation of PKB is often insufficient for tumorigenesis. Although initially identified as a weak oncogene, expression of PKB in most cells does not lead to transformation. Expression of membrane-targeted, myristylated PKB alleles can lead to oncogenic transformation [44,45], but this form of the kinase is activated well beyond the levels observed in tumors. In several cases, transgenic expression of activated alleles of PKB reduces the apoptotic potential of cells and promotes growth without inducing transformation; this raises the question of why this kinase is commonly activated in tumors. Transgenic expression of polyoma middle T antigen (PyMT) in mammary epithelial cells leads to aggressive tumor formation within 50–60 days. By its association with Src-family kinases, PyMT becomes tyrosine phosphorylated and recruits SHC and PI3K subunits by way of their phosphotyrosine binding domains. SHC couples to the activation of mouse Son of sevenless (mSOS) and Ras, whereas PI3K induces PKB and mTOR activation. MutaCurrent Opinion in Cell Biology 2005, 17:150–157
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tions of PyMT that selectively interfere with the recruitment of PI3K result in a significant delay in tumor formation that was also qualitatively distinct, being hyperplastic, low grade and riddled with apoptotic cells. Mammary-gland-specific, transgenic expression of an activated allele of PKB (in which the two activatory phosphorylation sites were replaced by aspartic acid, mimicking phosphorylation) has only a minor phenotype on its own in which involution of the gland following weaning is delayed by a few days. However, in mice expressing the mutant PyMT that is uncoupled from PI3K activation, co-expression of the activated PKB allele fully restores aggressive tumorigenesis [46]. Thus, PKB activation cooperates with and supports Ras activation, which by itself induces self-limiting apoptosis as well as growth. The human epidermal growth factor receptorlike 2 (HER2; also known as neu) receptor tyrosine kinase also activates the PI3K and Ras pathways and is directly implicated in breast cancer. Interestingly, expression of activated PKB decreases the latency of HER2/neu tumors [47]. Synergy between the activation of Ras and PKB might be manifested at the level of recruitment of mRNAs into polysomes, leading to efficient translation of mRNAs encoding growth-promoting proteins [48]. PKB activation is therefore likely to play a supportive role in tumorigenesis. By raising the threshold of pro-apoptotic signaling required for induction of apoptotic pathways and by increasing the metabolic capacity of cells, this kinase helps to suppress the normally suicide-inducing effects of DNA lesions and genomic instability, which typically occur during the transformation process. In essence, activation of PKB helps provide the ‘cancer cat’ with nine lives! Normal cells do not need this protection as their genomes are stable and they are receiving positive signals from the matrix. Only tumor cells ‘become addicted’ to chronic PKB activity. For this reason, this kinase should be a useful therapeutic target. One caveat might apply to the effect that activation of PKBa has on promoting differentiation. In the models of breast cancer that is driven by PyMT and HER2/neu, expression of PKB increased the rate of tumor growth but severely reduced the metastatic potential of the tumors. Tumors with reduced metastasis were found to be more highly differentiated [47]. This might have unwanted consequences for general PKB inhibitors as reduction of the kinase activity might increase the metastatic potential of otherwise localized lesions. In contrast to the reduction in metastasis observed by PKBa expression, overexpression of PKBb is associated with increased invasion and spread [49]. Thus, isoform-selective PKB inhibitors might prove to be very useful [50].
Glycogen synthase kinase-3 GSK-3 was the first physiological target of PKB to be identified and although other substrates might be more closely related to the role of PKB in oncogenesis, GSK-3 Current Opinion in Cell Biology 2005, 17:150–157
is a key player in PKB signaling [6]. There are two isoforms of GSK-3, termed a and b, which are encoded by distinct genes. As for PKB, there is some evidence for distinct, but overlapping, roles and each isoform is similarly regulated. PKB phosphorylates a serine residue within the N-terminal domain (Ser21 in GSK-3a and Ser9 in GSK-3b). This inhibits the kinase. The two isoforms are ubiquitously expressed and are highly conserved throughout eukaryotes. Indeed, clues to the physiological functions of this kinase have often derived from genetic analysis of simpler organisms. Unlike PKB, GSK3 is largely activated in resting, unstimulated cells. Its substrates are therefore highly phosphorylated in the absence of agonists. GSK-3 phosphorylates a wide array of proteins including metabolic enzymes, structural proteins and transcription factors. Where studied, the effect of GSK-3 phosphorylation tends to inactivate the substrate protein. Therefore, PKB-dependent inactivation of GSK-3 promotes the dephosphorylation and the activation of the many substrates of GSK-3 [7].
Priming site phosphorylation Many GSK-3 substrates share a peculiarity in that their phosphorylation by GSK-3 requires prior phosphorylation at a proximal residue by another protein kinase. The molecular basis of this unusual prerequisite for priming phosphorylation became apparent upon determination of the crystal structure of GSK-3b [51,52], as well as by some elegant mutagenesis [53]. Kinases related to GSK-3, such as cyclin-dependent kinases, p38 mitogen-activated protein kinases, and extracellular signal-dependent protein kinases, require phosphorylated residues on their T-loops loops for activity. A phosphothreonine and/or a phosphotyrosine aligns key b-strand and a-helical structures in the two lobes to allow entry of substrates to their catalytic sites. The T-loop of GSK-3 is phosphorylated at a single tyrosine (Tyr216 and Tyr279 for GSK-3b and GSK-3a, respectively). However, this site is highly phosphorylated (probably by an autophosphorylation mechanism [54]). In the crystal structure of GSK-3b, the conformation of the T-loop tyrosine does not directly preclude access to the ATP binding site when unphosphorylated, and phosphorylation only moderately increases activity. However, the relative positions of the N and C-terminal lobes of the kinase domain remain in an inactive conformation until the binding of a pre-phosphorylated substrate. The priming phosphate of the substrate binds to a positively charged pocket comprising of Arg96 and Arg180, as well as Lys205 (GSK-3b residues). This aligns the catalytic lobes into an optimal conformation for activity as well as orienting the substrate phospho-acceptor residue (Figure 2). The primed phosphate-binding pocket on GSK-3 serves another important regulatory function. Phosphorylation of the N-terminal domain at Ser21 or at Ser9 by PKB facilitates intramolecular binding of this residue to the www.sciencedirect.com
Protein kinase B signaling pathway Woodgett 155
Figure 2
(a) GSK-3 GSK-3 P Primed substrate
Substrate
P
P
GSK-3 P
P
Priming kinase
P Multi-site phosphorylation
(b) Primed phosphate docking pocket
PK(A, B or C) Ser9/21
P
NH2
P
GSK-3 Inhibitory kinase
Self-repressed Current Opinion in Cell Biology
Role of the phosphate-binding pocket of GSK-3 in substrate recognition and regulation. In (a) proteins only become substrates for GSK-3 upon phosphorylation by a priming kinase. GSK-3 binds to the phosphorylated substrate by way of a phosphate-binding pocket that causes an activating conformational change and properly aligns the substrate for phosphorylation. The phosphate introduced by GSK-3 can act as a priming site for subsequent phosphorylations in some targets (such as glycogen synthase and b-catenin). In (b) the phosphate-docking site can also bind to the intramolecular phosphate located at Ser9 (GSK-3b) or Ser21 (GSK-3a) that is introduced by other protein kinase (such as PKB) located within the N-terminal of GSK-3. This results in intramolecular occupation of the docking site by the N-terminal domain, precluding intermolecular binding to the primed phosphates of substrate proteins, effectively shutting down the enzyme.
substrate priming phosphate-binding pocket and by doing so, excludes binding of substrates, to effectively inhibit GSK-3 activity. In addition to PKB, several other protein kinases phosphorylate the inhibitory N-terminal site of GSK-3, including cyclic AMP-dependent kinase and certain PKCs (Figure 2) [55,56]. The priming phosphorylation site on substrates has a potential regulatory function as signals that modulate the degree of phosphorylation of these residues will affect their binding to GSK3. However, most priming sites are constitutively phosphorylated and are not signal dependent. In yeast and Dictyostelium, the substrates of the GSK-3 orthologues require priming but the kinases themselves do not harbour N-terminal regulatory phosphorylation sites, suggesting this level of regulation was a more recent event than substrate priming.
phosphate-binding pocket (PIF pocket) on PDK1 aids in its processing of the T-loop phosphorylation of PKB. The HM of PKB then assists in stabilizing an active conformation by intramolecular binding to the catalytic domain. A phosphate-binding pocket on GSK-3 directs it to specific substrates as well as providing a means of regulation through the intramolecular binding of a regulatory domain. Protein phosphorylation acts in this cascade as a selective binding mechanism that is analogous to Srchomology-2 (SH2) and phosphotyrosine binding (PTB)domain binding to phosphotyrosine at the level of receptor tyrosine kinases and their adapters. The remarkable degree of complexity in the regulation of this cascade, and of PKB in particular, is likely to reflect the dramatic consequences of its inappropriate activation as well as its core role in the engineered response to a variety of mitogens and growth factors.
Conclusions Analysis of the PDK/PKB/GSK-3 cascade reveals several recurring themes. Although these proteins couple to multiple pathways, their specific interactions are mediated through selective phosphate binding sites and recruitment to specific intracellular domains. The www.sciencedirect.com
Acknowledgements The author is supported by grants from the Canadian Institutes of Health Research and the Canadian Cancer Society, and is a Howard Hughes Medical Institute International Scholar. Current Opinion in Cell Biology 2005, 17:150–157
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