MARK Kinases in Cell Migration

CHAPTER SIX

Canonical and Noncanonical Roles of Par-1/MARK Kinases in Cell Migration Jocelyn A. McDonald1 Department of Molecular Genetics, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio, USA 1 Corresponding author: e-mail address: [email protected]

Contents 1. Introduction 2. Canonical Roles of Par-1/MARK in Cell Migration I: MTs 2.1 Par-1/MARK proteins 2.2 Regulation of MTs by Par-1/MARK 2.3 MT dynamics in directed cell migration 2.4 Par-1/MARK, MTs, and cell migration 3. Canonical Roles of Par-1/MARK in Cell Migration II: Cell Polarity 3.1 Cell polarity proteins in cell migration 3.2 Cell polarity and Par-1/MARK regulation of Drosophila border cell migration 3.3 Par-1/MARK and regulation of directional protrusions in migrating cells 4. Noncanonical Roles of Par-1/MARK in Cell Migration 4.1 Wnt pathways, Par-1/MARK, and cell movement during development 4.2 Par-1/MARK regulation of myosin during collective border cell migration 4.3 Role of Par-1/MARK in H. pylori CagA-dependent cell migration 5. Concluding Remarks Acknowledgments References

170 171 171 173 174 176 179 179 180 184 186 186 188 190 191 192 192

Abstract The partitioning defective gene 1 (Par-1)/microtubule affinity-regulating kinase (MARK) family of serine–threonine kinases have diverse cellular roles. Primary among these roles are the establishment and maintenance of cell polarity and the promotion of microtubule dynamics. Par-1/MARK kinases also regulate a growing number of cellular functions via noncanonical protein targets. Recent studies have demonstrated that Par-1/MARK proteins are required for the migration of multiple cell types. This review outlines the current evidence for regulation of cell migration by Par-1/MARK through both canonical and noncanonical roles. Par-1/MARK canonical control of microtubules during nonneuronal and neuronal migration is described. Next, regulation of cell polarity by Par-1/MARK and its dynamic effect on the movement of migrating cells are discussed.

International Review of Cell and Molecular Biology, Volume 312 ISSN 1937-6448 http://dx.doi.org/10.1016/B978-0-12-800178-3.00006-3

#

2014 Elsevier Inc. All rights reserved.

169

170

Jocelyn A. McDonald

As examples of recent research that have expanded, the roles of the Par-1/MARK in cell migration, noncanonical functions of Par-1/MARK in Wnt signaling and actomyosin dynamics are described. This review also highlights questions and current challenges to further understanding how the versatile Par-1/MARK proteins function in cell migration during development, homeostatic processes, and cancer.

1. INTRODUCTION The partitioning defective gene 1 (Par-1)/microtubule (MT) affinityregulating kinase (MARK) proteins belong to a family of serine–threonine kinases with roles in the establishment and maintenance of cell polarity and in the regulation of MTs. An elegant screen to identify genes required for the asymmetric division of the early Caenorhabditis elegans embryo originally isolated Par-1/MARK as one of a number of now-classic partitioning defective (Par) polarity proteins (Kemphues et al., 1988). In its capacity as a “polarity protein,” Par-1/MARK asymmetrically localizes to specific subcellular regions of cells (Goldstein and Macara, 2007). For example, Par-1/MARK is enriched at the posterior ends of Drosophila oocytes and C. elegans early embryos (Cox et al., 2001; Guo and Kemphues, 1995; Shulman et al., 2000). In epithelial cells, Par-1/MARK localizes basally or basolaterally and is specifically excluded from the apical domain (Fig. 6.1A; Shulman et al., 2000; Suzuki et al., 2004). Asymmetrically localized Par-1/MARK promotes the establishment of the basolateral (or posterior) domain of cells. Notably, human Par-1/MARK was independently identified as a regulator of MTs, based on its ability to phosphorylate and inhibit MT-associated proteins (MAPs) such as Tau (Drewes et al., 1997). The capacity of Par-1/ MARK proteins to promote cell polarity and to regulate stability and organization has each been well studied. Thus, these functions represent “canonical” roles (Fig. 6.1B). In the past decade, however, a number of studies have unearthed more complex roles for Par-1/MARK. These roles extend to new, unanticipated substrates and encompass alternate functions, depending on the cell type and/or specific context. Thus, Par-1/MARK has both canonical and noncanonical roles in cells. Accumulating evidence shows that Par-1/MARK regulates not only diverse types of cell movement, especially cell movement during development but also the movement or migration of cancer cells. In this review, I summarize our current understanding of the emerging functions of the Par-1/MARK kinase family in cell migration. I first provide an overview of how Par-1/MARK regulates MT dynamics and how this impacts both

171

Regulation of Cell Migration by Par-1/MARK

A

B Microtubules

aPKC (apical) Par-1 (basolateral)

Par-1

Polarity Noncanonical: Wnt signaling, myosin, etc.

C

PAR-1/MARK

08

95

T2

Human: PAR-1b/MARK2

N

Catalytic domain

T5

UBA

Linker/spacer

5

1 Fly: Par-1 identity (similarity)

KA1 74

88% (93%)

56% (87%)

Figure 6.1 Overview of canonical and noncanonical cellular roles of Par-1/MARK proteins. (A) Par-1/MARK is an asymmetrically localized protein. In epithelia, Par-1/MARK is enriched at basolateral membranes. Shown here is an example of Drosophila ovarian epithelial follicle cells stained for Par-1 (green; gray in the print version) and the apical protein aPKC (magenta; white in the print version); very little overlap of the proteins is observed. The apical and basal sides of the cells are at the top and bottom, respectively. (B) Known canonical (microtubules, cell polarity) and noncanonical roles of Par-1/MARK. (C) Schematic diagram of the Par-1/MARK family of proteins. Human Par-1b/MARK2 protein is shown. The fly Par-1 protein has similar organization and exhibits high homology within the catalytic and UBA domains (% identity/similarity is indicated). Two highly conserved phosphorylation sites are shown: the activating threonine 208 (black star) and the inactivating threonine 595 (white star). Abbreviations: N, N-terminal domain; UBA, ubiquitin-associated domain; KA1, kinase-associated 1 domain.

nonneuronal and neuronal cellular movements. Next, I describe newly emerging roles for Par-1/MARK as a cell polarity protein in cells that migrate during development. Subsequently, I highlight research from multiple model organisms that reveal the surprising complexity of noncanonical Par-1/MARK functions that promote Wnt signaling and actomyosin dynamics to regulate cell migration. Finally, I discuss some future directions and challenges that must be met to fully understand how Par-1/MARK functions in diverse cell movements during development, homeostatic processes, and in cancer.

2. CANONICAL ROLES OF PAR-1/MARK IN CELL MIGRATION I: MTs 2.1. Par-1/MARK proteins Par-1/MARK is a highly conserved member of the adenosine monophosphate-activated protein kinase (AMPK) family of serine–threonine

172

Jocelyn A. McDonald

kinases (Lizcano et al., 2004). Humans have four Par-1/MARK proteins: Par1a (MARK3), Par-1b (MARK2), Par-1c (MARK1), and Par-1d (MARK4). In contrast, Drosophila has a single par-1 gene, although this gene encodes up to 11 isoforms that range from short (357 amino acids) to long (827–1170 amino acids) variants. All Par-1/MARK homologs contain a well-conserved catalytic domain, an ubiquitin-associated (UBA) domain, a linker/spacer domain, and a kinase-associated 1 (KA1) domain (Fig. 6.1C). Human and fly Par-1/MARK isoforms primarily differ in the length of their divergent N-terminal and C-terminal linker/spacer domains. Several recent reviews describe the structural features of Par-1/MARK and how this kinase family is biochemically regulated in substantial detail (Marx et al., 2010; Matenia and Mandelkow, 2009; Naz et al., 2013). As a brief overview, most AMPK-related kinases, including PAR-1/MARK, have a UBA domain located adjacent to the catalytic kinase domain (Marx et al., 2010). In general, UBA domains have been extensively characterized as ubiquitin-binding protein modules. Interestingly, however, the Par-1/MARK UBA domain does not bind to ubiquitin with high affinity (Murphy et al., 2007). Instead, the UBA domain facilitates activation of Par-1/MARK catalytic domain by another kinase, LKB1 (also known as STK11) ( Jaleel et al., 2006; Rider, 2006). Other structural studies suggest that the UBA domain has an autoinhibitory function (Panneerselvam et al., 2006). Thus, it has been suggested that the UBA domain may act as an activation switch and/or stabilizing region (Marx et al., 2010; Murphy et al., 2007). Many Par-1/MARK isoforms (though not all) also have a KA1 domain, which helps Par-1 associate with the plasma membrane by binding phospholipids (Moravcevic et al., 2010). In Drosophila ovarian development, however, the KA1 domain appears to be functionally dispensable and Par-1 isoforms that lack this sequence are still able to localize correctly (Doerflinger et al., 2006; Huynh et al., 2001). Therefore, sequences other than the KA1 domain may also help to localize Par-1/MARK to appropriate membrane sites. Alternatively, there may be species-specific differences in how Par-1/MARK associates with the cell cortex. Further functional and biochemical studies are needed to clarify and reconcile these conflicting data on the specific roles of Par-1/MARK protein domains. Par-1/MARK is phosphorylated at multiple sites, although the relevance of many of these sites is unknown at present (G€ oransson et al., 2006). However, two phosphorylation sites are particularly relevant to the functions of Par-1/MARK proteins described in this review. First, all Par-1/MARK homologs are phosphorylated at a conserved site (T208 of Par-1b) in the catalytic domain. Phosphorylation at this site maximally activates kinase activity

Regulation of Cell Migration by Par-1/MARK

173

(Fig. 6.1C; Lizcano et al., 2004; Timm et al., 2003). Second, atypical Protein Kinase C (aPKC) phosphorylates a site within the divergent linker/spacer domain (T595 of Par-1b/MARK2) that is found across all eukaryotic species from yeast to humans (G€ oransson et al., 2006; Hurov et al., 2004; Kusakabe and Nishida, 2004). Phosphorylation at T595 blocks Par-1/MARK localization to the apical side of epithelia (Fig. 6.1C); this occurs through recruitment of 14-3-3 proteins, which regulate the subcellular distribution of Par-1/MARK (Hurov et al., 2004; Kusakabe and Nishida, 2004; Suzuki et al., 2004). Although one study found that phosphorylation by aPKC prevents Par-1/MARK kinase activity in cells (Hurov et al., 2004), other studies did not detect altered activity on in vitro substrates when T595 was mutated (G€ oransson et al., 2006; Kusakabe and Nishida, 2004). Nonetheless, phosphorylation of this site prevents apical recruitment and restricts Par-1/MARK to the basolateral domains of epithelia (Suzuki et al., 2004). Similarly, phosphorylation of this site restricts Par-1/MARK to the posterior of the Drosophila oocyte (Doerflinger et al., 2006; Vaccari et al., 2005).

2.2. Regulation of MTs by Par-1/MARK One of the most intensively investigated functions of Par-1/MARK is its highly conserved regulation of MTs. The MT cytoskeleton helps to polarize cells, organizes the cytoplasm, promotes intracellular trafficking and transport of vesicles, and participates in cell division (de Forges et al., 2012). MTs are polymers made up of repeating subunits of alpha- and beta-tubulin heterodimers (de Forges et al., 2012; Desai and Mitchison, 1997). The orientation of guanosine triphosphate (GTP)-bound tubulin in these hollow tubes gives MTs a specific polarity within the cell. MT minus ends are typically anchored to a centrosome or MT-organizing center (MTOC). In contrast, the MT plus ends are more dynamic and exhibit more rapid exchange of tubulin monomers. As a result, MTs grow primarily from their plus ends, extending in the direction of the cell periphery or plasma membrane. The cycles of dynamic growth and retraction/disassembly of the MT polymers have been termed “dynamic instability” (Desai and Mitchison, 1997). Subsets of MTs exhibit greater stability and persistent growth. Both posttranslational modifications of tubulin (e.g., acetylation) and association of tubulin with different MAPs have been shown to stabilize such MTs (de Forges et al., 2012). Par-1/MARK proteins control MT dynamics primarily through phosphorylation of MAPs, MT-specific proteins that regulate the growth,

174

Jocelyn A. McDonald

stability, cross-linking, and collapse of MTs. In particular, Par-1/MARK phosphorylates a conserved Lys-X-Gly-Ser (KXGS; X is any amino acid) motif that is found in multiple MAPs (Drewes et al., 1997). Phosphorylation by Par-1/MARK negatively regulates the MAPs Tau, MAP2, and MAP4, each of which normally promotes MT stability. Phosphorylation causes these MAPs to dissociate from MTs and in turn destabilize MTs (Drewes et al., 1997). Moreover, Tau phosphorylated by Par-1/MARK is associated with increased levels of neurofibrillary tangles in Alzheimer’s disease (Chin et al., 2000; Mandelkow and Mandelkow, 2012). In surprising contrast to results obtained from mammalian cells, studies carried out in Drosophila suggest that Par-1 stabilizes MTs (Doerflinger et al., 2003). These contrasting findings suggest that there are species and/or cell differences with respect to how Par-1/MARK modulates MTs. To address this, Hayashi and colleagues directly measured MT plus-end growth in live neuronal dendrites in which Par-1b/MARK2 function was impaired (Hayashi et al., 2011). Par-1b/MARK2 knockdown decreased MT growth but did not affect the levels of polymerized MTs (Hayashi et al., 2011, 2012). Hayashi and coworkers propose that Par-1/MARK proteins increases MT dynamic instability (net growth) rather than simply promoting the polymerization/ stabilization of MTs. Taken together, these studies demonstrate that Par-1/MARK kinases have highly conserved roles in the regulation of MTs in a variety of cell types.

2.3. MT dynamics in directed cell migration One of the most important roles for dynamic MTs is to promote directed cell migration. Movement of various cell types is integral to embryonic development, immune system function, wound healing, and other homeostatic processes. Dysregulated cell migration contributes to inflammation, birth defects, and cancer. For example, cells that are normally stationary can lose their epithelial characteristics and become migratory during tumor invasion and metastasis. The process by which cells move, whether normal or abnormal, has been termed as the cell motility cycle (Ridley et al., 2003). To move forward, cells undergo successive rounds of protrusion at the front of the cell, dynamic adhesion with the migratory substrate (other cells or extracellular matrix), and retraction of the cell rear (Fig. 6.2A; Ridley et al., 2003). Migrating cells typically respond to chemoattractant gradients and signals within tissues, which induce cell polarization and influence cell movement trajectories (Lara Rodriguez and Schneider, 2013; Reig et al.,

175

Regulation of Cell Migration by Par-1/MARK

A

Par-1/MARK and MTs in nonneuronal cell migration Front

Nucleus Migration Rear

[

Centrosome (MTOC) Microtubules

Actin-rich lamellipodium

MARK2 downstream of Rac1: • Organize pioneer MTs • MT dynamics • Polarized cell shape • Leading edge protrusion

Pioneer microtubules

B Par-1/MARK in neuronal cell migration Wild type MARK2 shRNA

x

CP IZ SVZ

Radial glia

VZ

Figure 6.2 Role of Par-1/MARK in microtubule (MT) regulation and cell migration. (A) Nonneuronal cell migration. Schematic of a generic migrating cell showing the oriented distribution of microtubules from the MTOC primarily toward the front or leading edge. Par-1b/MARK2 functions downstream of Rac1 to regulate microtubules during migration (Nishimura et al., 2012). (B) Diagram of Par-1/MARK regulation of radial neuronal cell migration during development of the cerebral cortex. In wild-type brains (left side of the diagram), neuroblasts in the ventricular zone (VZ) differentiate, become multipolar (subventricular zone, SVZ), change to a bipolar morphology and migrate along the radial glia (intermediate zone, IZ). These neurons continue migrating until they reach the cortical plate (CP), where they extend axons into the marginal zone (top). In brains treated with Par-1b/MARK2 shRNA by in utero electroporation (right side of the diagram), many neurons remain at the multipolar stage. Those bipolar neurons that do form generally stall in the IZ with abnormal morphology and do not reach the CP (Sapir et al., 2008a).

2014). Regulation of the cellular cytoskeleton is absolutely essential for all cells that migrate. F-actin and associated regulatory proteins provide mechanical structure and microscale forces to drive extension and retraction of protrusions and lamellae. Dynamic MTs also play crucial, albeit complex and more poorly understood, roles in cell migration, some of which may depend on the given type of cell (Etienne-Manneville, 2013; Kaverina and Straube, 2011).

176

Jocelyn A. McDonald

Well-known roles for MTs in cell migration include polarization of cells, trafficking of vesicles and organelles, and modulation of adhesions (EtienneManneville, 2013; Small et al., 2002; Stehbens and Wittmann, 2012; Watanabe et al., 2005). MTs are anchored from their minus ends at the MTOC, which is located just in front of or behind the nucleus depending on the cell. MT plus ends are typically oriented toward the cell periphery (Fig. 6.2A). Migrating cells exhibit a characteristic asymmetric distribution of MTs. This asymmetric distribution arises through stabilization of MTs at the front and destabilization toward the back by respective regulatory proteins (Kaverina and Straube, 2011). Thus, MT growth is generally directed toward the leading edge in migrating cells (Fig. 6.2A). Most MTs do not enter the actin-rich lamellipodium at the very front edge of the cell, in which F-actin undergoes retrograde flow (Fig. 6.2A; Etienne-Manneville, 2013; Waterman-Storer and Salmon, 1997). However, a relative minority of slow-growing “pioneer” MTs enter this region to promote cell membrane protrusion and subsequent migration (Waterman-Storer and Salmon, 1997). The small GTPase Rac1 stabilizes and promotes growth of these pioneer MTs (Wittmann et al., 2003). Interestingly, some cells (e.g., fish keratocytes) do not require MTs for movement (Kaverina and Straube, 2011). However, larger cell size correlates with a greater need for MTs in cell migration. The requirement for MTs in a migrating cell is also influenced by the cohort of MAPs and other MT-regulatory proteins present in the cell. In addition, most cells require both F-actin and MTs for directed cell migration (Etienne-Manneville, 2013).

2.4. Par-1/MARK, MTs, and cell migration 2.4.1 Nonneuronal cell migration Given the known roles for Par-1/MARK in the regulation of MT stability and dynamics, it is not surprising that Par-1/MARK influences cell migration by regulating MTs. Indeed, a recent study implicates Par-1b/MARK2 in polarization of MTs via the Rac1 GTPase in nonneuronal cell migration (Nishimura et al., 2012). Rac1 promotes pioneer MTs at the leading edge of migrating cells (Wittmann et al., 2003). This MT-dependent role for Rac1 supplements its known role in the promotion of F-actin-dependent protrusion extension (Ridley, 2011). In a screen to find specific MT regulators downstream of Rac1 GTPase in leading edge protrusions, Nishimura et al. (2012) identified Par-1b/MARK2. Constitutively active (CA)-Rac1 produces very stable MTs; this causes cells to become round with broad lamellipodia and MTs are misoriented, such that they grow parallel to the cell

Regulation of Cell Migration by Par-1/MARK

177

edge rather than growing perpendicular to the cell membrane (Fig. 6.2A; Nishimura et al., 2012; Wittmann et al., 2003). The identity of MT regulators that produce pioneer MTs in response to Rac1 activation was unclear. Therefore, Nishimura et al. (2012) knocked down 23 different MT-regulatory proteins in human U2-OS osteosarcoma cells expressing CA-Rac1 and measured MT dynamics using automated tracking. Only Par-1b/MARK2 knockdown rescued multiple phenotypes caused by CA-Rac1—cell shape, MT dynamics, and MT orientation defects. In particular, knockdown of Par-1b/MARK2 suppressed Rac1-induced MTs parallel to the cell membrane. Thus, Par-1b/MARK2 functions downstream of Rac1 in promoting lamellipodial protrusion. How does Par-1/MARK regulate leading edge MTs and how does this impact cell migration? Knockdown of Par-1b/MARK2 in U2-OS cells altered MT growth dynamics (Nishimura et al., 2012). However, in contrast to its effect in dendrites (Section 2.2), Par-1b/MARK2 slowed MT growth in migrating U2-OS cells (Hayashi et al., 2011, 2012; Nishimura et al., 2012). These differences in regulation of MTs by Par-1/MARK may be due to differing expression levels of specific MAPs in different cell types. Nonetheless, this study reveals that Par-1/MARK regulation of MTs is important for cell migration. Specifically, knockdown of Par-1b/MARK2 disrupted cell migration in a wound-healing assay (Nishimura et al., 2012). Cells with reduced Par-1b/MARK2 exhibited slower migration, misoriented centrosomes, and MTs that no longer extended into the leading edge. Par-1b/MARK2 knock-down cells also exhibited reduced directional persistence, a measure of how well and consistently cells move in a specific direction. Based on these data, Nishimura et al. (2012) propose that Par-1b/MARK promotes directional cell migration and cell polarity by regulating MTs downstream of Rac1 (Fig. 6.2A). As will be described below, this model fits well with the role for Par-1/MARK in neuronal migration (Section 2.4.2). 2.4.2 Neuronal cell migration During brain development, neuronal cells undergo a dramatic migration to populate the growing brain. Neuroblasts (neuronal precursor cells) differentiate and move out of the ventricular zone (VZ) to become multipolar neurons (Fig. 6.2B; Kriegstein and Noctor, 2004; Noctor et al., 2001; Reiner and Sapir, 2014). These neurons then transition to become bipolar, move along radial glia, and migrate until they reach the cortical plate, their final position within the cerebral cortex (Fig. 6.2B; Kriegstein and Noctor,

178

Jocelyn A. McDonald

2004). Interestingly, Par-1/MARK is required for neuronal cell polarity (Biernat et al., 2002; Chen et al., 2006; Shelly and Poo, 2011). Moreover, mice that are mutant for Par-1b/MARK2 display learning impairments (Segu et al., 2008). Such observations suggested that Par-1/MARK has a role in brain development. To test this idea, Sapir et al. (2008a) examined how Par-b/MARK2 influences the developing mouse brain. Sapir et al. (2008a) performed in utero electroporation to target Par-1b/MARK2 knockdown (or perform overexpression) specifically in the brain. Remarkably, knockdown of Par-1b/MARK2 caused neuronal cells to halt their migration at the intermediate zone (Fig. 6.2B). These neurons exhibited abnormal centrosome dynamics, with altered coupling of the centrosome to the nucleus (Sapir et al., 2008a). Many neurons did not transition from the multipolar stage, thus revealing a role for Par-1b/MARK2 in the polarization of migrating neurons (Fig. 6.2B). Interestingly, even those neurons that became bipolar exhibited abnormal morphology (Fig. 6.2B; Sapir et al., 2008a). Overexpression of Par-1b/MARK2 also prevented neuronal migration; the cells became round and failed to migrate (Sapir et al., 2008a). This result indicates that precise levels of Par-1b/MARK2 activity are required for efficient migration during brain development. Moreover, Par-1b/ MARK2 is needed for the directed migration of postnatal neuroblasts in the olfactory bulb (Mejia-Gervacio et al., 2012). These and other examples suggest that Par-1/MARK is a key regulator of neuronal migration at various stages of development. The importance of Par-1b/MARK2 in neuronal migration is at least partly due to its regulation of MT dynamics (Reiner and Sapir, 2014). As expected from studies carried out in other cell types (see Sections 2.2 and 2.4.1), knockdown of Par-1b/MARK2 in primary neurons altered MT stability and dynamics (Sapir et al., 2008a). Moreover, during the migration of cortical neurons, Par-1b/MARK2 acts in opposition to Doublecortin (DCX), a MAP that stabilizes MTs (Sapir et al., 2008b). This is significant because Par-1b/MARK2 phosphorylates DCX and prevents DCX binding to MTs (Schaar et al., 2004). Sapir et al. (2008b) found that simultaneous reduction of Par-1b/MARK2 and DCX partially suppresses the cortical migration defect caused by DCX knockdown alone. In addition, Par-1b/ MARK2 and DCX colocalize in olfactory bulb neuroblasts (MejiaGervacio et al., 2012). It is important to note, however, that while these studies demonstrate MT-dependent roles for Par-1/MARK in neuronal migration, they do not definitively show that all of Par-1/MARK’s functions are mediated through MTs. Thus, further studies are needed to address

Regulation of Cell Migration by Par-1/MARK

179

whether and to what extent Par-1/MARK proteins have MT-independent roles in neuronal migration and brain development.

3. CANONICAL ROLES OF PAR-1/MARK IN CELL MIGRATION II: CELL POLARITY 3.1. Cell polarity proteins in cell migration The second canonical role for Par-1/MARK proteins is to promote the polarization of many different types of cells. While members of the Par family of proteins were first discovered in C. elegans, these proteins are recognized as major regulators of cell polarity in most eukaryotic cells (Goldstein and Macara, 2007; Kemphues et al., 1988). Par proteins regulate apical–basal polarity of eukaryotic epithelial cells and establish the anterior–posterior polarity of Drosophila oocytes and early C. elegans embryos (Goldstein and Macara, 2007). Moreover, in mammals, all of the Par proteins are required for the polarization of neurons during axon outgrowth (Shelly and Poo, 2011). Par-3 and Par-6 are scaffold proteins that generally form a complex with aPKC; this Par–aPKC complex localizes to the apical side of epithelia (Fig. 6.1A). In contrast, Par-1 localizes to the basolateral side (Fig. 6.1A). Several other protein complexes—the apical Crumbs (Crb) complex (Crb/Pals1/Patj) and the basolateral Scribble (Scrib) complex (Dlg/Scrib/ Lgl)—are also required for epithelial polarity and interact in various ways with the Par polarity proteins (Rodriguez-Boulan and Macara, 2014; Roignot et al., 2013). The regulation of polarity in migrating cells by Par-1/MARK is less well understood (see Section 3.2); however, most of the other known epithelial cell polarity proteins are implicated in the regulation of directional migration of a range of cell types. In migrating cells, polarity proteins localize to the front or leading edge and promote polarized migration (Etienne-Manneville and Hall, 2001; Goldstein and Macara, 2007; Pinheiro and Montell, 2004; Shin et al., 2007). The apically localized Par-6/aPKC proteins regulate the directional migration of astrocytes by orienting MTs and the MTOC (EtienneManneville and Hall, 2001). A similar requirement for Par-3 in centrosome orientation was identified in migrating NIH-3T3 cells (Schmoranzer et al., 2009). Moreover, Par-6 and aPKC function downstream of the small GTPase Cdc42 in the polarization of migrating astrocytes and fibroblasts (Cau and Hall, 2005; Etienne-Manneville and Hall, 2001; Joberty et al., 2000). Cdc42 and other members of the Rho family GTPases (e.g., Rac1 and RhoA) organize the F-actin cytoskeleton in migrating cells (Hall,

180

Jocelyn A. McDonald

2012; Ridley, 2011). The apical protein Patj (part of the Crb complex) localizes to the cell front where it recruits Par-3 and aPKC, leading to efficient migration of epithelial cells (Shin et al., 2007). Surprisingly, the Scrib complex, which is basolaterally localized in epithelia, also localizes to the leading edge of migrating cells where it promotes cell polarization and directed migration (Dow et al., 2007; Etienne-Manneville, 2008; EtienneManneville et al., 2005; Osmani et al., 2006). Although the apical Par–aPKC and basolateral Scrib complex proteins each localize to the front of migrating cells, it is unclear whether and to what extent these proteins interact with each other during cell migration.

3.2. Cell polarity and Par-1/MARK regulation of Drosophila border cell migration 3.2.1 Cell polarity and the border cell model of collective migration Cell polarity proteins are absolutely required for the migration of Drosophila border cells, a genetically tractable in vivo model of cell migration. Border cells are a group of 6–10 cells in the ovary that undergo a specific type of cell movement, called “collective migration,” in which cells remain associated with each other and move as a unit (Friedl and Gilmour, 2009; Montell et al., 2012). Other examples of collective movements include the chain-like migration of neural crest cells, sheet movement of tissues during embryonic gastrulation, and cohort-type migration in tumor invasion and metastasis (Friedl and Gilmour, 2009). In the case of border cells, 4–8 cells that are initially part of the follicle cell epithelium are recruited by a pair of polar cells to form a cluster (Fig. 6.3A). Once specified, border cells detach as a group from the follicular epithelium and migrate between the germline-derived nurse cells. Border cells migrate 100 μm until they reach the oocyte where they later contribute to formation of the micropyle, a structure in the egg shell that functions as the sperm-entry site for oocyte fertilization (Montell et al., 1992, 2012). Like all collectively migrating cells, border cells maintain high levels of adhesion between cells to establish cluster cohesion and integrity. Apical cell polarity complexes play a critical role in organizing and maintaining the border cell cluster during their movement. Par-3, Par-6, and aPKC initially localize to the front of the border cell cluster prior to their detachment from the epithelium (Pinheiro and Montell, 2004). However, once border cells leave the epithelium, the characteristic epithelial apical–basal polarity is altered (Montell et al., 2012; Niewiadomska et al., 1999; Pinheiro and Montell, 2004). At this stage, border cells undergo a rotation whereby

181

Regulation of Cell Migration by Par-1/MARK

A

Premigration (detachment) eS9 NC

NC

NC

Migration

Migration is complete S9

S10

Oocyte

NC

NC

NC

Border cells (BC)

BC Follicle cell (fc) epithelium

BC

pc pc

BC

BC

BC

Migration

B

D Control

GFP PC DAPI

par-1 mutant

Oocyte Wild type

C

BC

E

Par-1 in Detachment

1 Polarity High Par-1

Par-1 in directional migration 2 Myosin

{

Par-3

High Par-1 fc

pc pc

{

Model 1: Par-1 promotes stable protrusions in leading cells

= localized fc decrease or fc remodeling of adhesions

fc Additional MyoP kinase(s)

{

pMRLC High myo-II

Par-1 pc pc fc fc

= localized contraction

pc Lead BC pc

Model 2: Par-1 inhibits stable protrusions in nonleading cells

Figure 6.3 Overview of Drosophila border cell migration and roles for Par-1 in this process. (A) Schematic of border cell migration during ovarian egg chamber development. Border cells (dark gray) develop from the somatic follicle cell epithelium starting at early stage 9 (eS9; left panel), detach from the epithelium and migrate between the nurse cells (NC) at S9 (middle panel) and complete their migration to the oocyte (light gray) by S10 (right panel). An expanded view of the border cell cluster is shown (middle panel); 4–8 border cells (BC) surround a central pair of polar cells (PC; light gray). (B) Micrographs showing S10 egg chambers with mosaic mutant cells marked by GFP (green; light gray in the print version), polar cells (PC, red; dark gray in the print version), and DNA stained with DAPI (blue; dark gray in the print version). Left panel, wild-type border cells (arrow) migrate and reach the oocyte at the correct stage. Right panel, border cells mutant for par-1 (arrow) stay at the tip of the egg chamber connected to mutant follicle cells. (C) Schematic models of Par-1 function during detachment of border cells from the follicular epithelium. Par-1 (dotted lines) localizes to the basolateral/rear of the cluster at the stage of detachment. Model 1 (polarity) shows the role of Par-1 in cell polarity regulation (McDonald et al., 2008). Model 2 (myosin) depicts the role of Par-1 in regulation of myo-II activity (pMRLC) via myosin phosphatase (MyoP) (Majumder et al., 2012). (D) Stills from live time-lapse movies of control (left panel) and par-1 mutant (right panel) border cell clusters. Control border cells generally have a single stable protrusion (arrow) at the front in the direction of migration. par-1 mutant border cell clusters often have misdirected protrusions in addition to those that form at the front (arrows). (E) Model for basolateral Par-1 in directional border cell protrusions. In model 1, Par-1 (dotted line) does not localize to the front of the cluster and instead promotes the localization of an unknown protein to the lead border cell (gray); this stabilizes protrusions at the front. In model 2, Par-1 destabilizes or inhibits protrusions from nonleading border cells, either directly or indirectly on unknown target proteins. Anterior is to the left in all panels. The image in panel E is adapted from Majumder et al. (2012).

182

Jocelyn A. McDonald

the polarity proteins now localize perpendicular to the direction of migration. Moreover, high levels of Par–aPKC complex proteins are expressed at the interfaces between border cells, coincident with cell adhesion proteins such as E-cadherin (Niewiadomska et al., 1999; Pinheiro and Montell, 2004). Loss of par-3 or par-6 severely disrupts border cell migration (Pinheiro and Montell, 2004). Most border cells mutant for par-3 or par-6 (or both) stop migrating before they reach the oocyte. Interestingly, however, the organization of these border cell clusters is severely disrupted and characterized by pulling away or extrusion of mutant border cells from the rest of the cluster (Llense and Martı´n-Blanco, 2008; Pinheiro and Montell, 2004). Localization of Par-3, Par-6, and cell adhesion proteins is disrupted in border cells mutant for the Jun-kinase pathway (JNK). JNK mutants display a remarkably similar cluster cohesion defect to that observed upon loss of the Par–aPKC complex, and overexpression of Par-3 is able to suppress the phenotype (Llense and Martı´n-Blanco, 2008). Moreover, just as in astrocytes and fibroblasts, Cdc42 is also involved. Thus, Par–aPKC promotes cluster cohesion via Cdc42 and JNK signaling, likely because these proteins promote adhesion between cells (Hidalgo-Carcedo et al., 2011; Llense and Martı´n-Blanco, 2008; Rodriguez-Boulan and Macara, 2014). This idea is supported by recent studies carried out in cancer cells, which find that the Par–aPKC complex maintains cell contacts in migrating collectives and prevents individual cell invasion and metastasis (Hidalgo-Carcedo et al., 2011; McCaffrey et al., 2012; Xue et al., 2013). Other polarity complexes are also required for border cell migration, although their functions are still poorly understood and have not been intensively investigated. A recent screen for additional proteins that regulate border cell migration identified a requirement for the Crb complex in border cell migration (Aranjuez et al., 2012). Moreover, Crb and associated proteins (Pals1, Patj, and Veli/Lin-7) have a similar distribution as the Par–aPKC complex proteins (Aranjuez et al., 2012; Niewiadomska et al., 1999; Pinheiro and Montell, 2004; Tanentzapf et al., 2000). Further work is needed to determine if the Crb complex has comparable functions to the Par–aPKC apical complex in border cells. The basolateral Scrib complex appears to suppress rather than promote border cell migration, in contrast to its role in human astrocytes and epithelial cells (see Section 3.1). Specifically, loss of the Scrib complex members dlg1 and lgl cause follicle cells to overproliferate and form motile streams that invade the nurse cells (Goode and Perrimon, 1997; Szafranski and Goode, 2007). Loss of lgl disrupts the

Regulation of Cell Migration by Par-1/MARK

183

cohesiveness of the border cell cluster, suggesting that the Scrib complex may function later during border cell migration (Li et al., 2011). As with the Crb complex, further investigation is needed to fully understand the function for the Scrib complex in border cells. As described further below (Section 3.2.2), Par-1/MARK also localizes to the basolateral domain of epithelia and border cells. In contrast to the Scrib complex, however, Par-1/MARK promotes the detachment and directional migration of border cells (McDonald et al., 2008). 3.2.2 Role of Par-1 in border cell migration and polarity Despite the fact that the apical Par–aPKC complex is critically required for proper border cell migration, Par-1/MARK was not implicated in border cell migration until it was isolated from a forward-genetic screen. Multiple mutagenesis screens have been performed to identify genes and molecular pathways required for border cell migration in an unbiased manner (Bai et al., 2000; Liu and Montell, 1999; Mathieu et al., 2007; Silver and Montell, 2001). From one of these screens, a mutant allele was isolated that severely disrupted border cell movement (Liu and Montell, 1999). A second mutagenesis screen isolated additional noncomplementing alleles, which facilitated classical genetic mapping to a single gene locus, par-1 (McDonald et al., 2008). Further analysis showed that loss of par-1 prevented many border cells from detaching and moving away from the anterior follicle cell epithelium (Fig. 6.3B; McDonald et al., 2008). Specifically, Par-1 is required in both border cells and adjacent follicle cells to promote border cell detachment from the epithelium prior to migration (Fig. 6.3A–C). Consistent with its localization in nonmigratory epithelial cells, Par-1 localizes to the basolateral side of the border cell cluster prior to its detachment from the epithelium (Fig. 6.3C). Moreover, at detachment stages, the Par-1 protein localization domain does not overlap with that of the apical Par-3 and aPKC proteins. Clues to how Par-1 promotes border cell detachment came from its known roles in the regulation of cell polarity. In epithelia, Par-1/MARK phosphorylates several conserved 14-3-3-binding sites in Par-3 (Benton and St Johnston, 2003; Hurd et al., 2003). 14-3-3 (also known as Par-5 in its capacity as another Par polarity protein) is a scaffolding protein that can shuttle proteins to different cellular compartments and/or alter conformation of target proteins (Bridges and Moorhead, 2005). Phosphorylation of Par-3 by Par-1/MARK promotes binding of 14-3-3 to Par-3, which prevents Par-3 localization at the basolateral membrane and restricts it to the

184

Jocelyn A. McDonald

apical domain (Benton and St Johnston, 2003; Hurd et al., 2003). In border cells, loss of par-1 causes Par-3 and the cell adhesion protein E-cadherin to be mislocalized and/or upregulated between border cells and the adjacent epithelial follicle cells (McDonald et al., 2008). Failure of border cells to leave the epithelium in par-1 mutants is likely due to improper persistence of adhesion and polarity between border cells and follicle cells (Fig. 6.3B and C). Importantly, border cell detachment and migration are strongly inhibited when the 14-3-3-binding sites of Par-3 are mutated and in mutants for leonardo, the Drosophila 14-3-3ζ homolog. Therefore Par-1, through 14-3-3-dependent inhibition of Par-3 localization, promotes the localized decrease and/or remodeling of adhesions between border cells and follicle cells (Fig. 6.3C). Par-1/MARK proteins also have a role in cell movement during gastrulation of Xenopus embryos that requires binding to 14-3-3 protein and phosphorylation by aPKC (Kusakabe and Nishida, 2004). These findings together demonstrate that Par-1/MARK has conserved roles in promoting diverse cell migrations through its canonical role as a cell polarity protein.

3.3. Par-1/MARK and regulation of directional protrusions in migrating cells Par-1 has a second important function in border cells—promotion of directional protrusions. Border cells, like all migrating cells, have a leading edge or front. One or two border cells at the front of the cluster extend (and retract) protrusions, which help the cluster as a whole to navigate and move toward the oocyte (Fulga and Rørth, 2002; Prasad and Montell, 2007; Wang et al., 2010). While Par-1 clearly has roles during detachment, many par-1 mutant border cells that are able to detach from the epithelium, nevertheless, do not complete their migration to the oocyte (McDonald et al., 2008). Live imaging analyses further revealed that par-1-deficient border cells exhibit a significant number of misoriented protrusions (Fig. 6.3D). Importantly, the effect of Par-1 on protrusion direction is independent of Par-3. There are two possible mechanisms for regulation of protrusions by Par-1. First, Par-1 promotes the localization of a factor to the front of the cluster that stabilizes protrusions specifically in the leading border cells (Fig. 6.3E). Alternatively, Par-1 specifically inhibits or destabilizes protrusions from nonleading border cells (Fig. 6.3E). In support of the latter model, uniform overexpression of Par-1 in all border cells significantly disrupted protrusion formation, resulting in short protrusions that rapidly retracted (K. Sawant and J.A. McDonald, unpublished). Further work, however, is

Regulation of Cell Migration by Par-1/MARK

185

needed to determine the precise mechanism for how Par-1 regulates directional protrusions. A hint as to how Par-1 may function in protrusions comes from studies on how border cells respond to guidance signaling. In border cells, the small GTPase Rac promotes F-actin-rich protrusions in response to two receptor tyrosine kinases (RTKs), the epidermal growth factor receptor and the platelet-derived growth factor/vascular endothelial growth factor receptor related (Duchek and Rørth, 2001; Duchek et al., 2001; McDonald et al., 2006). Ligands for each of these RTKs are secreted by the oocyte and act as attractants to guide border cells along their migratory path (Duchek et al., 2001; McDonald et al., 2003, 2006). The polarized extension of protrusions depends absolutely on RTK activation; loss of both RTKs results in misdirected protrusions and stalled border cell migration (Prasad and Montell, 2007). Moreover, localized Rac activation is necessary and sufficient for a single lead border cell to extend dynamic protrusions (Wang et al., 2010). The misdirected protrusions caused by loss of par-1 strongly resemble those observed in border cells in which both RTKs are inhibited (McDonald et al., 2008; Prasad and Montell, 2007). Moreover, we recently identified a genetic interaction between par-1 and Rac during border cell migration (Geisbrecht et al., 2013). As described earlier (Section 2.4.1), Par-1b/MARK2 is required for Rac1-dependent MT growth dynamics in migrating human cells (Nishimura et al., 2012). While these studies suggest a possible mechanistic connection between Drosophila Par-1 and Rac, loss of par-1 did not visibly alter the spatial distribution of MTs in border cells (McDonald et al., 2008). However, neither the dynamics nor the polarity of MTs was assessed in par-1 mutant border cells. Therefore, it will be essential in the future to determine whether (and to what extent) Par-1 is required downstream of RTK signaling for Rac-dependent polarized protrusions via MT dynamics. The molecular target(s) of Par-1 in protrusion stability, whether MT-dependent or not, also remains to be identified. The requirement for Par-1/MARK in mammalian cell migration, apart from its roles in MT dynamics (see Section 2.4), has been poorly studied. A potential reason for this lack of understanding is that the four human Par-1/MARK homologous proteins may exhibit substantial functional redundancy, which would make it difficult to determine how essential each homologue is for cell migration. A number of polarity complexes promote centrosome orientation during directed cell movement of human cells (see Section 3.1). Similarly, knockdown of Par-1b/MARK2 in human osteosarcoma cells causes misoriented centrosomes and inefficient migration

186

Jocelyn A. McDonald

(Nishimura et al., 2012). However, Drosophila border cells do not have apparent centrosomes or MTOCs, but nevertheless require Par-1 and the apical Par–aPKC proteins for their directed migration (McDonald et al., 2008; Pinheiro and Montell, 2004; Van de Bor et al., 2011; Yang et al., 2012). This raises the interesting possibility that human Par-1/MARK proteins promote cell migration by organizing cell polarity without necessarily acting through MT dynamics or through orientation of centrosomes/ MTOCs.

4. NONCANONICAL ROLES OF PAR-1/MARK IN CELL MIGRATION As a family of serine–threonine kinases, Par-1/MARK proteins phosphorylate a growing number of identified protein substrates outside of the canonical MAPs (Section 2) and Par polarity proteins (Section 3). These targets include proteins involved in the cell cycle (Cdc25), synapse formation (Dlg1), an E3 ubiquitin ligase (RNF41) involved in basement membrane deposition of laminin, mitochondrial transport (PTEN-induced kinase 1; PINK1), and many others (Hurov and Piwnica-Worms, 2007; Lewandowski and Piwnica-Worms, 2014; Matenia and Mandelkow, 2009, 2014; Matenia et al., 2012; Peng et al., 1998; Zhang et al., 2007). Such noncanonical roles reflect the diversity (and growing list) of cellular functions regulated by the well-conserved Par-1/MARK kinases. Three noncanonical functions of Par-1/MARK involved in cell movements are of particular interest—regulation of Wnt signaling pathways (Section 4.1), regulation of nonmuscle myosin II activity (Section 4.2), and Helicobactor pylori CagA protein-dependent cell migration (Section 4.3).

4.1. Wnt pathways, Par-1/MARK, and cell movement during development One surprising role for Par-1/MARK was its identification as a regulator of the Wnt pathway member Dishevelled (Dvl). Dvl is an essential scaffolding protein that mediates both canonical and noncanonical Wnt signaling (Wallingford and Habas, 2005). The latter has also been termed planar cell polarity (PCP). In canonical Wnt signaling, Dvl scaffolds multiple signaling components to activate β-catenin, which in turn translocates to the nucleus and acts as a transcriptional coactivator of target genes. In the PCP pathway, Dvl forms a complex with proteins that organize the F-actin cytoskeleton,

Regulation of Cell Migration by Par-1/MARK

187

independent of β-catenin activation. PCP proteins establish the planar polarity of cells, perpendicular to the apical–basal axis, within tissues. Par-1/MARK was identified as a Dvl-associated kinase required for canonical Wnt signaling (Sun et al., 2001). However, other studies suggested that Par-1/MARK was required for gastrulation of Xenopus embryos but did not affect transcription of canonical Wnt targets (Kusakabe and Nishida, 2004). The convergent extension movements that occur during gastrulation require PCP signaling (Munoz-Soriano et al., 2012). These seemingly conflicting findings were resolved when it was discovered that different Par-1b/ MARK2 protein isoforms mediate canonical and noncanonical Wnt pathways (Ossipova et al., 2005). Knockdown of the Par-1b/MARK2 “X” isoform, as well as Par-1a/MARK3, disrupted early dorsoanterior development of Xenopus embryos, a canonical Wnt-dependent process. Moreover, loss of Par-1a/MARK3 or Par-1bX/MARK2X reduced the expression of canonical Wnt transcriptional targets required for specification of the dorsal organizer. In contrast, knockdown of the longer Par-1b/ MARK2 “Y” isoform caused later embryonic defects in convergent extension but did not affect canonical Wnt pathway activation (Ossipova et al., 2005). Translocation of Dvl to the cell membrane is required for PCP signaling (Axelrod et al., 1998; Wallingford and Habas, 2005). Importantly, Par-1bY/MARK2Y promotes Dvl membrane localization through phosphorylation of previously identified sites in Dvl (Ossipova et al., 2005; Sun et al., 2001). However, mutation of these Par-1/MARKphosphorylation sites did not affect the ability of Dvl to activate the canonical Wnt pathway (Ossipova et al., 2005). Thus, different Par-1/MARK isoforms control different branches of the Wnt signaling pathway during embryonic development. The requirement for Par-1/MARK in noncanonical Wnt signaling appears to be relevant to other migrating cells. Transforming growth factor-beta (TGFβ) signaling stimulates epithelial-to-mesenchymal transitions (EMT) that occur in various developmental processes and in cancer (Massague´, 2012; Thiery et al., 2009). While there is known cross talk between TGFβ and canonical Wnt pathways in EMT, Mamidi et al. (2012) found that Par-1/MARK connects noncanonical Wnt signaling to TGFβ. Specifically, Par-1b/MARK2 promotes cell migration during wound healing in response to TGFβ activation (Mamidi et al., 2012). Moreover, regulation of cell migration by Par-1b/MARK2 depends on Dvl and the noncanonical Wnt-5a ligand (Mamidi et al., 2012). PCP genes including Drosophila dvl (dsh) are also required for border cell migration (Bastock and

188

Jocelyn A. McDonald

Strutt, 2007). Nonetheless, the phenotypes caused by loss of PCP genes are distinct from par-1, suggesting that Dvl is not a major target of Par-1 in border cell migration (Bastock and Strutt, 2007; McDonald et al., 2008). Therefore, additional studies are needed to determine the extent to which Par-1/MARK regulates noncanonical Wnt pathways during the migration of other cells, either during development or in cancer.

4.2. Par-1/MARK regulation of myosin during collective border cell migration As described earlier (Section 3.2.2), Par-1 is required for the collective detachment and migration of Drosophila border cells. While the previous study showed that Par-1 negatively regulated Par-3 to remodel adhesions and promote migration, not all Par-1 functions are mediated through regulation of Par-3 (McDonald et al., 2008). For example, Par-1 promotes directional protrusion independent of Par-3. Moreover, maintenance of properly polarized distributions of Par-1 versus Par-3 at follicle cell-border cell junctions is unlikely to be the exclusive mechanism needed for detachment. In a search for additional targets of Par-1 during border cell detachment and migration, Majumder et al. (2012) found that Par-1 promotes activation of nonmuscle myosin II (myo-II) through regulation of myosin phosphatase (MyoP). Myo-II is a major regulator of cell migration, cell shape, and tissue morphogenesis (Gorfinkiel and Blanchard, 2011; Munjal and Lecuit, 2014; Vicente-Manzanares et al., 2009). Myo-II is a multicomponent protein complex consisting of dimers of each of three proteins: a heavy chain (MHC), an essential light chain, and a regulatory light chain (MRLC). Activation of myo-II primarily occurs through phosphorylation of MRLC at two amino acids—mammalian threonine 18 and serine 19 (T18S19; Drosophila T20S21). Conversely, dephosphorylation of T18S19 by MyoP inactivates myo-II. During cell migration, actin and myo-II (actomyosin) contractile networks help to establish cell polarity, modulate cell adhesion, and retract the cell rear (Vicente-Manzanares et al., 2007, 2009). Additionally, actomyosin contraction can regulate the retraction of leading edge protrusions (Shutova et al., 2012; Vicente-Manzanares et al., 2009). Previous studies showed a requirement for myo-II in border cell migration (Edwards and Kiehart, 1996; Fulga and Rørth, 2002). Loss of myo-II using mutant alleles of the Drosophila MRLC, spaghetti squash (sqh), resulted in abnormally lengthened and irregularly shaped border cell protrusions (Fulga and Rørth, 2002). Moreover, disruption of myo-II by knockdown

Regulation of Cell Migration by Par-1/MARK

189

of MRLC or expression of a dominant-negative version of MHC inhibited border cell detachment from the epithelium (Majumder et al., 2012). These studies indicated that myo-II has multiple roles in border cells—detachment from the epithelium, retraction of leading edge protrusions, and retraction of the cluster rear—all of which promote efficient migration. Remarkably, par-1 mutants also exhibited long, misshapen leading edge protrusions in addition to the detachment and migration defects described earlier (Section 3.2.2) (McDonald et al., 2008). The similarity of phenotypes caused by loss of myo-II and par-1 suggested that Par-1 may target actomyosin contraction. To test this hypothesis, Majumder et al. (2012) performed a series of genetic tests between par-1 and myo-II. Simultaneous knockdown of both Par-1 and MRLC by RNAi inhibited border cell migration more strongly than knockdown of the individual genes (Majumder et al., 2012). In addition, phosphomimetic mutant MRLC transgenes (in which T18S19 was mutated E18E19) strongly suppressed the detachment and migration defects caused by Par-1 knockdown. These genetic interactions indicated a close link between active myo-II and Par-1. In live border cells, myo-II (visualized by MRLC-GFP) is highly dynamic and accumulates in punctate fluorescent spots (foci) near cell membranes. Myo-II foci have been observed in other epithelia and correlate with high levels of actomyosin contraction (Gorfinkiel and Blanchard, 2011; He et al., 2010; Martin et al., 2009; Rauzi et al., 2010). Live myo-II foci were lost when Par-1 was knocked down, suggesting that Par-1 regulates myo-II protein dynamics in border cells (Majumder et al., 2012). Moreover, levels of phosphorylated MRLC were reduced in par-1 mutant border cells and increased when Par-1 was overexpressed. These observations together indicated that Par-1 functions upstream of active myo-II in border cells. Although Par-1 could directly phosphorylate MRLC to influence myo-II activity, Majumder et al. (2012) instead found that Par-1 is present in a protein complex with MyoP in cells. MyoP consists of two proteins, a protein phosphatase 1 catalytic subunit and a substrate-specific myosinbinding subunit (MBS; also known as MYPT1). Phosphorylation of MBS at several sites including T696 (Drosophila T594) inhibits MyoP activity (Grassie et al., 2011; Kimura et al., 1996). Levels of phosphorylated T696 were decreased when Par-1 was knocked down in ovaries. Moreover, purified Par-1 kinase was capable of phosphorylating MBS at T696 in in vitro kinase assays. Thus, Par-1 phosphorylation of MBS inhibits MyoP. Interestingly, Par-1 is enriched at the basolateral side (rear) of the border cell cluster,

190

Jocelyn A. McDonald

whereas MyoP is uniformly distributed (Majumder et al., 2012; McDonald et al., 2008). Thus, asymmetric Par-1 locally inactivates MyoP resulting in increased levels of active myo-II at the cluster rear. Such localized actomyosin contraction promotes border cell detachment from the epithelium and retraction of the cluster rear (Fig. 6.3C). Par-1/MARK may only regulate myo-II activation in specific contexts. For example, in contrast to border cells, Par-1 does not colocalize with either MyoP or myo-II in epithelial follicle cells—Par-1 is basolateral, whereas MyoP and myo-II are apical in epithelia (P. Majumder and J.A. McDonald, unpublished) (Shulman et al., 2000; Wang and Riechmann, 2007). While myo-II and Par-1 both regulate protrusions at the front of the border cell cluster, Par-1 does not generally localize to the front, at least prior to detachment (Fulga and Rørth, 2002; Majumder et al., 2012; McDonald et al., 2008). Therefore, other kinases likely activate myo-II throughout the border cell cluster. Rho-associated kinase (ROCK; Drosophila Rok) is a likely candidate, as it can directly phosphorylate MRLC (Amano et al., 1996, 2010). Significantly, rok mutants have lower levels of phosphorylated MRLC and defective border cell migration (G. Aranjuez, P. Majumder, and J.A. McDonald, unpublished) (Majumder et al., 2012). Surprisingly, in mammalian epithelial MDCK cells, Par-1b/MARK2 promotes lateral “hepatic-type” lumen polarity by inhibiting myo-II activity (Cohen et al., 2007; La´zaro-Die´guez et al., 2013). Thus, while Par1/MARK proteins are connected to myo-II in multiple cell types, there may be species- and/or cell type differences as to whether Par-1/MARK positively or negatively regulates myo-II activity.

4.3. Role of Par-1/MARK in H. pylori CagA-dependent cell migration Par-1/MARK plays another key noncanonical role in the shape and migration of cells transformed by H. pylori. H. pylori bacterial infection causes chronic gastritis, ulcers, and gastric adenocarcinomas (Yamahashi and Hatakeyama, 2013). The most virulent strains of H. pylori encode the CagA protein, which transform cells from a columnar epithelial shape to an elongated shape, the so-called hummingbird phenotype (Saadat et al., 2007). One of the host proteins that CagA targets in cells is Par-1/MARK (Saadat et al., 2007; Yamahashi and Hatakeyama, 2013). Binding of CagA inactivates Par-1/MARK kinase activity (Lu et al., 2009; Saadat et al., 2007). Inhibition of Par-1/MARK subsequently alters cell morphology in a CagA-dependent manner. Wild-type CagA protein increased the speed

Regulation of Cell Migration by Par-1/MARK

191

of cell migration; however, a mutant CagA protein that cannot bind Par1/MARK suppressed this cell motility. These results indicate that CagA binding to (and inhibition of ) Par-1/MARK promotes cell migration of epithelial cells (Kikuchi et al., 2012). Cells overexpressing Par-1/MARK have altered F-actin cytoskeletal distribution (Lu et al., 2009). The small GTPase RhoA promotes actin organization in both stationary and migratory cells (Hall, 2012; Ridley, 2011). This observation prompted Yamahashi and colleagues to test a role for Par-1/MARK proteins on RhoA activity. Indeed, Par-1b/MARK2 phosphorylates and inhibits an activator of the RhoA GTPase, the guanine nucleotide exchange factor GEF-H1 (Yamahashi et al., 2011). Through inhibition of GEF-H1, Par-1b/MARK2 prevents the formation of RhoA-dependent F-actin stress fibers in cells. Taking these studies together, Yamahashi and Hatakeyama (2013) propose that CagA inactivation of Par-1/MARK leads to RhoA activation, which in turn promotes cell shape changes and cell motility. Thus, CagA-dependent inhibition of Par-1/ MARK stimulates cell migration. However, this role for Par-1/MARK is opposite to what has been found in other cells where active Par-1/MARK promotes migration (see earlier sections). These differences could be due to different molecular pathways that function downstream of the CagA–Par-1/ MARK complex in gastric epithelia. More work is needed, however, to fully understand the differences between normal cell migration and migration that is induced by CagA-positive H. pylori infection. Future studies on the interactions between CagA and Par-1/MARK may provide new insights into how cells undergo morphological transformation in cancer.

5. CONCLUDING REMARKS Par-1/MARK kinases have complex roles in diverse examples of cell migration. On the one hand, Par-1/MARK supports cell migration through canonical regulation of MT dynamics and cell polarity. On the other hand, Par-1/MARK regulates noncanonical substrates in a variety of moving cells. Answering several key questions will help us better understand the functions of Par-1/MARK proteins in cell migration and motility. First, to what extent are the four human Par-1/MARK proteins redundant with each other? Most of the studies identifying roles for Par-1/MARK in cell migration have focused specifically on Par-1b/MARK2. Systematic co-knockdown of multiple Par-1/MARK proteins (and/or splice isoforms) may reveal more widespread roles in cell motility. Second, are there general

192

Jocelyn A. McDonald

functions for Par-1/MARK in cell migration, or do Par-1/MARK proteins have specific functions depending on the cell type? At present, the studies on Par-1/MARK-mediated noncanonical regulation of Wnt signaling and myo-II activity via MyoP appear to have uncovered cell-specific roles. However, these models have not yet been tested in other tissues and organisms. Thus, this is an open and interesting question that remains to be addressed. Third, are there other noncanonical targets of Par-1/MARK involved in regulating cell migration? Several recent studies implicate Par-1/MARK kinase activity in the Hippo tissue growth pathway both in Drosophila epithelia and human cells (Huang et al., 2013; Mohseni et al., 2013). The Hippo pathway is required for border cell detachment and migration (Lucas et al., 2013). Thus, it will be important to determine if Par-1 targets Hippo activity in both border cells and other migrating cells. Finally, do human Par-1/MARK homologs participate in tumor invasion and metastasis? Expression of Par-1/MARK proteins has been found to be elevated in glioblastoma, squamous cell carcinoma, and prostate cancer tissues (Beghini et al., 2003; Huang et al., 2013; Magnani et al., 2011; Roversi et al., 2005; Sun et al., 2004). These data suggest the intriguing possibility that downregulation of Par-1/MARK activities could be a therapeutic target in some tumors. In the coming years, it will be exciting to discover new roles for Par-1/MARK in the migration of normal cells and in cancer.

ACKNOWLEDGMENTS I would like to thank Saurav Misra for helpful comments on the chapter. Work in the McDonald lab is supported by funds from the Cleveland Clinic.

REFERENCES Amano, M., Ito, M., Kimura, K., Fukata, Y., Chihara, K., Nakano, T., Matsuura, Y., Kaibuchi, K., 1996. Phosphorylation and activation of myosin by Rho-associated kinase (Rho-kinase). J. Biol. Chem. 271, 20246–20249. Amano, M., Nakayama, M., Kaibuchi, K., 2010. Rho-kinase/ROCK: a key regulator of the cytoskeleton and cell polarity. Cytoskeleton 67, 545–554. Aranjuez, G., Kudlaty, E., Longworth, M.S., McDonald, J.A., 2012. On the role of PDZ domain-encoding genes in Drosophila border cell migration. G3: Genes Genomes Genet. 2, 1379–1391. Axelrod, J.D., Miller, J.R., Shulman, J.M., Moon, R.T., Perrimon, N., 1998. Differential recruitment of Dishevelled provides signaling specificity in the planar cell polarity and Wingless signaling pathways. Genes Dev. 12, 2610–2622. Bai, J., Uehara, Y., Montell, D.J., 2000. Regulation of invasive cell behavior by Taiman, a Drosophila protein related to AIB1, a steroid receptor coactivator amplified in breast cancer. Cell 103, 1047–1058.

Regulation of Cell Migration by Par-1/MARK

193

Bastock, R., Strutt, D., 2007. The planar polarity pathway promotes coordinated cell migration during Drosophila oogenesis. Development 134, 3055–3064. Beghini, A., Magnani, I., Roversi, G., Piepoli, T., Di Terlizzi, S., Moroni, R.F., Pollo, B., Conti, A.M.F., Cowell, J.K., Finocchiaro, G., Larizza, L., 2003. The neural progenitor-restricted isoform of the MARK4 gene in 19q13.2 is upregulated in human gliomas and overexpressed in a subset of glioblastoma cell lines. Oncogene 22, 2581–2591. Benton, R., St Johnston, D., 2003. Drosophila PAR-1 and 14-3-3 inhibit Bazooka/PAR-3 to establish complementary cortical domains in polarized cells. Cell 115, 691–704. Biernat, J., Wu, Y.-Z., Timm, T., Zheng-Fischh€ ofer, Q., Mandelkow, E., Meijer, L., Mandelkow, E.-M., 2002. Protein kinase MARK/PAR-1 is required for neurite outgrowth and establishment of neuronal polarity. Mol. Biol. Cell 13, 4013–4028. Bridges, D., Moorhead, G.B.G., 2005. 14-3-3 Proteins: a number of functions for a numbered protein. Sci. STKE 2005, re10. Cau, J., Hall, A., 2005. Cdc42 controls the polarity of the actin and microtubule cytoskeletons through two distinct signal transduction pathways. J. Cell Sci. 118, 2579–2587. Chen, Y.M., Wang, Q.J., Hu, H.S., Yu, P.C., Zhu, J., Drewes, G., Piwnica-Worms, H., Luo, Z.G., 2006. Microtubule affinity-regulating kinase 2 functions downstream of the PAR-3/PAR-6/atypical PKC complex in regulating hippocampal neuronal polarity. Proc. Natl. Acad. Sci. U.S.A. 103, 8534–8539. Chin, J.Y., Knowles, R.B., Schneider, A., Drewes, G., Mandelkow, E.-M., Hyman, B.T., 2000. Microtubule-affinity regulating kinase (MARK) is tightly associated with neurofibrillary tangles in Alzheimer brain: a fluorescence resonance energy transfer study. J. Neuropathol. Exp. Neurol. 59, 966–971. Cohen, D., Tian, Y., Mu¨sch, A., 2007. Par1b promotes hepatic-type lumen polarity in Madin Darby canine kidney cells via myosin II- and E-cadherin-dependent signaling. Mol. Biol. Cell 18, 2203–2215. Cox, D.N., Lu, B., Sun, T.Q., Williams, L.T., Jan, Y.N., 2001. Drosophila par-1 is required for oocyte differentiation and microtubule organization. Curr. Biol. 11, 75–87. de Forges, H., Bouissou, A., Perez, F., 2012. Interplay between microtubule dynamics and intracellular organization. Int. J. Biochem. Cell Biol. 44, 266–274. Desai, A., Mitchison, T.J., 1997. Microtubule polymerization dynamics. Annu. Rev. Cell Dev. Biol. 13, 83–117. Doerflinger, H., Benton, R., Shulman, J.M., St Johnston, D., 2003. The role of PAR-1 in regulating the polarised microtubule cytoskeleton in the Drosophila follicular epithelium. Development 130, 3965–3975. Doerflinger, H., Benton, R., Torres, I.L., Zwart, M.F., St Johnston, D., 2006. Drosophila anterior-posterior polarity requires actin-dependent PAR-1 recruitment to the oocyte posterior. Curr. Biol. 16, 1090–1095. Dow, L.E., Kauffman, J.S., Caddy, J., Zarbalis, K., Peterson, A.S., Jane, S.M., Russell, S.M., Humbert, P.O., 2007. The tumour-suppressor Scribble dictates cell polarity during directed epithelial migration: regulation of Rho GTPase recruitment to the leading edge. Oncogene 26, 2272–2282. Drewes, G., Ebneth, A., Preuss, U., Mandelkow, E.M., Mandelkow, E., 1997. MARK, a novel family of protein kinases that phosphorylate microtubule-associated proteins and trigger microtubule disruption. Cell 89, 297–308. Duchek, P., Rørth, P., 2001. Guidance of cell migration by EGF receptor signaling during Drosophila oogenesis. Science 291, 131–133. Duchek, P., Somogyi, K., Je´kely, G., Beccari, S., Rørth, P., 2001. Guidance of cell migration by the Drosophila PDGF/VEGF receptor. Cell 107, 17–26. Edwards, K.A., Kiehart, D.P., 1996. Drosophila nonmuscle myosin II has multiple essential roles in imaginal disc and egg chamber morphogenesis. Development 122, 1499–1511.

194

Jocelyn A. McDonald

Etienne-Manneville, S., 2008. Polarity proteins in migration and invasion. Oncogene 27, 6970–6980. Etienne-Manneville, S., 2013. Microtubules in cell migration. Annu. Rev. Cell Dev. Biol. 29, 471–499. Etienne-Manneville, S., Hall, A., 2001. Integrin-mediated activation of Cdc42 controls cell polarity in migrating astrocytes through PKCζ. Cell 106, 489–498. Etienne-Manneville, S., Manneville, J.-B., Nicholls, S., Ferenczi, M.A., Hall, A., 2005. Cdc42 and Par6-PKCzeta regulate the spatially localized association of Dlg1 and APC to control cell polarization. J. Cell Biol. 170, 895–901. Friedl, P., Gilmour, D., 2009. Collective cell migration in morphogenesis, regeneration and cancer. Nat. Rev. Mol. Cell Biol. 10, 445–457. Fulga, T.A., Rørth, P., 2002. Invasive cell migration is initiated by guided growth of long cellular extensions. Nat. Cell Biol. 4, 715–719. Geisbrecht, E.R., Sawant, K., Su, Y., Liu, Z.C., Silver, D.L., Burtscher, A., Wang, X., Zhu, A.J., McDonald, J.A., 2013. Genetic interaction screens identify a role for hedgehog signaling in Drosophila border cell migration. Dev. Dyn. 242, 414–431. Goldstein, B., Macara, I.G., 2007. The PAR proteins: fundamental players in animal cell polarization. Dev. Cell 13, 609–622. Goode, S., Perrimon, N., 1997. Inhibition of patterned cell shape change and cell invasion by Discs large during Drosophila oogenesis. Genes Dev. 11, 2532–2544. G€ oransson, O., Deak, M., Wullschleger, S., Morrice, N.A., Prescott, A.R., Alessi, D.R., 2006. Regulation of the polarity kinases PAR-1/MARK by 14-3-3 interaction and phosphorylation. J. Cell Sci. 119, 4059–4070. Gorfinkiel, N., Blanchard, G.B., 2011. Dynamics of actomyosin contractile activity during epithelial morphogenesis. Curr. Opin. Cell Biol. 23, 531–539. Grassie, M.E., Moffat, L.D., Walsh, M.P., MacDonald, J.A., 2011. The myosin phosphatase targeting protein (MYPT) family: a regulated mechanism for achieving substrate specificity of the catalytic subunit of protein phosphatase type 1δ. Arch. Biochem. Biophys. 510, 147–159. Guo, S., Kemphues, K.J., 1995. par-1, a gene required for establishing polarity in C. elegans embryos, encodes a putative Ser/Thr kinase that is asymmetrically distributed. Cell 81, 611–620. Hall, A., 2012. Rho family GTPases. Biochem. Soc. Trans. 40, 1378–1382. Hayashi, K., Suzuki, A., Hirai, S.-I., Kurihara, Y., Hoogenraad, C.C., Ohno, S., 2011. Maintenance of dendritic spine morphology by partitioning-defective 1b through regulation of microtubule growth. J. Neurosci. 31, 12094–12103. Hayashi, K., Suzuki, A., Ohno, S., 2012. PAR-1/MARK: a kinase essential for maintaining the dynamic state of microtubules. Cell Struct. Funct. 37, 21–25. He, L., Wang, X., Tang, H.L., Montell, D.J., 2010. Tissue elongation requires oscillating contractions of a basal actomyosin network. Nat. Cell Biol. 12, 1133–1142. Hidalgo-Carcedo, C., Hooper, S., Chaudhry, S.I., Williamson, P., Harrington, K., Leitinger, B., Sahai, E., 2011. Collective cell migration requires suppression of actomyosin at cell-cell contacts mediated by DDR1 and the cell polarity regulators Par3 and Par6. Nat. Cell Biol. 13, 49–58. Huang, H.-L., Wang, S., Yin, M.-X., Dong, L., Wang, C., Wu, W., Lu, Y., Feng, M., Dai, C., Guo, X., Li, L., Zhao, B., Zhou, Z., Ji, H., Jiang, J., Zhao, Y., Liu, X.-Y., Zhang, L., 2013. Par-1 regulates tissue growth by influencing hippo phosphorylation status and hippo-salvador association. PLoS Biol. 11, e1001620. Hurd, T.W., Fan, S., Liu, C.J., Kweon, H.K., Hakansson, K., Margolis, B., 2003. Phosphorylation-dependent binding of 14-3-3 to the polarity protein Par3 regulates cell polarity in mammalian epithelia. Curr. Biol. 13, 2082–2090.

Regulation of Cell Migration by Par-1/MARK

195

Hurov, J., Piwnica-Worms, H., 2007. The Par-1/MARK family of protein kinases: from polarity to metabolism. Cell Cycle 6, 1966–1969. Hurov, J.B., Watkins, J.L., Piwnica-Worms, H., 2004. Atypical PKC phosphorylates PAR-1 kinases to regulate localization and activity. Curr. Biol. 14, 736–741. Huynh, J.R., Shulman, J.M., Benton, R., St Johnston, D., 2001. PAR-1 is required for the maintenance of oocyte fate in Drosophila. Development 128, 1201–1209. Jaleel, M., Villa, F., Deak, M., Toth, R., Prescott, A.R., Van Aalten, D.M.F., Alessi, D.R., 2006. The ubiquitin-associated domain of AMPK-related kinases regulates conformation and LKB1-mediated phosphorylation and activation. Biochem. J. 394, 545–555. Joberty, G., Petersen, C., Gao, L., Macara, I.G., 2000. The cell-polarity protein Par6 links Par3 and atypical protein kinase C to Cdc42. Nat. Cell Biol. 2, 531–539. Kaverina, I., Straube, A., 2011. Regulation of cell migration by dynamic microtubules. Semin. Cell Dev. Biol. 22, 968–974. Kemphues, K.J., Priess, J.R., Morton, D.G., Cheng, N., 1988. Identification of genes required for cytoplasmic localization in early C. elegans embryos. Cell 52, 311–320. Kikuchi, K., Murata-Kamiya, N., Kondo, S., Hatakeyama, M., 2012. Helicobacter pylori stimulates epithelial cell migration via CagA-mediated perturbation of host cell signaling. Microbes Infect. 14, 470–476. Kimura, K., Ito, M., Amano, M., Chihara, K., Fukata, Y., Nakafuku, M., Yamamori, B., Feng, J., Nakano, T., Okawa, K., Iwamatsu, A., Kaibuchi, K., 1996. Regulation of myosin phosphatase by Rho and Rho-associated kinase (Rho-kinase). Science 273, 245–248. Kriegstein, A.R., Noctor, S.C., 2004. Patterns of neuronal migration in the embryonic cortex. Trends Neurosci. 27, 392–399. Kusakabe, M., Nishida, E., 2004. The polarity-inducing kinase Par-1 controls Xenopus gastrulation in cooperation with 14-3-3 and aPKC. EMBO J. 23, 4190–4201. Lara Rodriguez, L., Schneider, I.C., 2013. Directed cell migration in multi-cue environments. Integr. Biol. 5, 1306–1323. La´zaro-Die´guez, F., Cohen, D., Fernandez, D., Hodgson, L., van IJzendoorn, S.C.D., Mu¨sch, A., 2013. Par1b links lumen polarity with LGN-NuMA positioning for distinct epithelial cell division phenotypes. J. Cell Biol. 203, 251–264. Lewandowski, K.T., Piwnica-Worms, H., 2014. Phosphorylation of the E3 ubiquitin ligase RNF41 by the kinase Par-1b is required for epithelial cell polarity. J. Cell Sci. 127, 315–327. Li, Q., Feng, S., Yu, L., Zhao, G., Li, M., 2011. Requirements of Lgl in cell differentiation and motility during Drosophila ovarian follicular epithelium morphogenesis. Fly 5, 81–87. Liu, Y., Montell, D.J., 1999. Identification of mutations that cause cell migration defects in mosaic clones. Development 126, 1869–1878. Lizcano, J.M., G€ oransson, O., Toth, R., Deak, M., Morrice, N.A., Boudeau, J., Hawley, S.A., Udd, L., Ma¨kela¨, T.P., Hardie, D.G., Alessi, D.R., 2004. LKB1 is a master kinase that activates 13 kinases of the AMPK subfamily, including MARK/PAR-1. EMBO J. 23, 833–843. Llense, F., Martı´n-Blanco, E., 2008. JNK signaling controls border cell cluster integrity and collective cell migration. Curr. Biol. 18, 538–544. Lu, H., Murata-Kamiya, N., Saito, Y., Hatakeyama, M., 2009. Role of partitioningdefective 1/microtubule affinity-regulating kinases in the morphogenetic activity of Helicobacter pylori CagA. J. Biol. Chem. 284, 23024–23036. Lucas, E.P., Khanal, I., Gaspar, P., Fletcher, G.C., Polesello, C., Tapon, N., Thompson, B.J., 2013. The Hippo pathway polarizes the actin cytoskeleton during collective migration of Drosophila border cells. J. Cell Biol. 201, 875–885. Magnani, I., Novielli, C., Fontana, L., Tabano, S., Rovina, D., Moroni, R.F., Bauer, D., Mazzoleni, S., Colombo, E.A., Tedeschi, G., Monti, L., Porta, G., Bosari, S.,

196

Jocelyn A. McDonald

Frassoni, C., Galli, R., Bello, L., Larizza, L., 2011. Differential signature of the centrosomal MARK4 isoforms in glioma. Anal. Cell. Pathol. 34, 319–338. Majumder, P., Aranjuez, G., Amick, J., McDonald, J.A., 2012. Par-1 controls myosin-II activity through myosin phosphatase to regulate border cell migration. Curr. Biol. 22, 363–372. Mamidi, A., Inui, M., Manfrin, A., Soligo, S., Enzo, E., Aragona, M., Cordenonsi, M., Wessely, O., Dupont, S., Piccolo, S., 2012. Signaling crosstalk between TGFβ and Dishevelled/Par1b. Cell Death Differ. 19, 1689–1697. Mandelkow, E.-M., Mandelkow, E., 2012. Biochemistry and cell biology of tau protein in neurofibrillary degeneration. Cold Spring Harb. Perspect. Med. 2, a006247. Martin, A.C., Kaschube, M., Wieschaus, E.F., 2009. Pulsed contractions of an actin-myosin network drive apical constriction. Nature 457, 495–499. Marx, A., Nugoor, C., Panneerselvam, S., Mandelkow, E., 2010. Structure and function of polarity-inducing kinase family MARK/Par-1 within the branch of AMPK/Snf1related kinases. FASEB J. 24, 1637–1648. Massague´, J., 2012. TGFβ signalling in context. Nat. Rev. Mol. Cell Biol. 13, 616–630. Matenia, D., Mandelkow, E.-M., 2009. The tau of MARK: a polarized view of the cytoskeleton. Trends Biochem. Sci. 34, 332–342. Matenia, D., Mandelkow, E.M., 2014. Emerging modes of PINK1 signaling: another task for MARK2. Front. Mol. Neurosci. 7, 37. Matenia, D., Hempp, C., Timm, T., Eikhof, A., Mandelkow, E.M., 2012. Microtubule affinity-regulating kinase 2 (MARK2) turns on phosphatase and tensin homolog (PTEN)-induced kinase 1 (PINK1) at Thr-313, a mutation site in parkinson disease: effects on mitochondrial transport. J. Biol. Chem. 287, 8174–8186. Mathieu, J., Sung, H.-H., Pugieux, C., Soetaert, J., Rørth, P., 2007. A sensitized PiggyBacbased screen for regulators of border cell migration in Drosophila. Genetics 176, 1579–1590. McCaffrey, L.M., Montalbano, J., Mihai, C., Macara, I.G., 2012. Loss of the Par3 polarity protein promotes breast tumorigenesis and metastasis. Cancer Cell 22, 601–614. McDonald, J.A., Pinheiro, E.M., Montell, D.J., 2003. PVF1, a PDGF/VEGF homolog, is sufficient to guide border cells and interacts genetically with Taiman. Development 130, 3469–3478. McDonald, J.A., Pinheiro, E.M., Kadlec, L., Schupbach, T., Montell, D.J., 2006. Multiple EGFR ligands participate in guiding migrating border cells. Dev. Biol. 296, 94–103. McDonald, J.A., Khodyakova, A., Aranjuez, G., Dudley, C., Montell, D.J., 2008. PAR-1 kinase regulates epithelial detachment and directional protrusion of migrating border cells. Curr. Biol. 18, 1659–1667. Mejia-Gervacio, S., Murray, K., Sapir, T., Belvindrah, R., Reiner, O., Lledo, P.-M., 2012. MARK2/Par-1 guides the directionality of neuroblasts migrating to the olfactory bulb. Mol. Cell. Neurosci. 49, 97–103. Mohseni, M., Sun, J., Lau, A., Curtis, S., Goldsmith, J., Fox, V.L., Wei, C., Frazier, M., Samson, O., Wong, K.-K., Wong, K.-K., Kim, C., Camargo, F.D., 2013. A genetic screen identifies an LKB1-MARK signalling axis controlling the Hippo-YAP pathway. Nat. Cell Biol. 16, 108–117. Montell, D.J., Rørth, P., Spradling, A.C., 1992. slow border cells, a locus required for a developmentally regulated cell migration during oogenesis, encodes Drosophila CEBP. Cell 71, 51–62. Montell, D.J., Yoon, W.H., Starz-Gaiano, M., 2012. Group choreography: mechanisms orchestrating the collective movement of border cells. Nat. Rev. Mol. Cell Biol. 13, 631–645.

Regulation of Cell Migration by Par-1/MARK

197

Moravcevic, K., Mendrola, J.M., Schmitz, K.R., Wang, Y.-H., Slochower, D., Janmey, P.A., Lemmon, M.A., 2010. Kinase associated-1 domains drive MARK/PAR1 kinases to membrane targets by binding acidic phospholipids. Cell 143, 966–977. Munjal, A., Lecuit, T., 2014. Actomyosin networks and tissue morphogenesis. Development 141, 1789–1793. Munoz-Soriano, V., Belacortu, Y., Paricio, N., 2012. Planar cell polarity signaling in collective cell movements during morphogenesis and disease. Curr. Genomics 13, 609–622. Murphy, J.M., Korzhnev, D.M., Ceccarelli, D.F., Briant, D.J., Zarrine-Afsar, A., Sicheri, F., Kay, L.E., Pawson, T., 2007. Conformational instability of the MARK3 UBA domain compromises ubiquitin recognition and promotes interaction with the adjacent kinase domain. Proc. Natl. Acad. Sci. U.S.A. 104, 14336–14341. Naz, F., Anjum, F., Islam, A., Ahmad, F., Hassan, M.I., 2013. Microtubule affinity-regulating kinase 4: structure, function, and regulation. Cell Biochem. Biophys. 67, 485–499. Niewiadomska, P., Godt, D., Tepass, U., 1999. DE-Cadherin is required for intercellular motility during Drosophila oogenesis. J. Cell Biol. 144, 533–547. Nishimura, Y., Applegate, K., Davidson, M.W., Danuser, G., Waterman, C.M., 2012. Automated screening of microtubule growth dynamics identifies MARK2 as a regulator of leading edge microtubules downstream of Rac1 in migrating cells. PLoS One 7, e41413. Noctor, S.C., Flint, A.C., Weissman, T.A., Dammerman, R.S., Kriegstein, A.R., 2001. Neurons derived from radial glial cells establish radial units in neocortex. Nature 409, 714–720. Osmani, N., Vitale, N., Borg, J.-P., Etienne-Manneville, S., 2006. Scrib controls Cdc42 localization and activity to promote cell polarization during astrocyte migration. Curr. Biol. 16, 2395–2405. Ossipova, O., Dhawan, S., Sokol, S., Green, J.B., 2005. Distinct PAR-1 proteins function in different branches of Wnt signaling during vertebrate development. Dev. Cell 8, 829–841. Panneerselvam, S., Marx, A., Mandelkow, E.-M., Mandelkow, E., 2006. Structure of the catalytic and ubiquitin-associated domains of the protein kinase MARK/Par-1. Structure 14, 173–183. Peng, C.Y., Graves, P.R., Ogg, S., Thoma, R.S., Byrnes 3., M.J., Wu, Z., Stephenson, M.T., Piwnica-Worms, H., 1998. C-TAK1 protein kinase phosphorylates human Cdc25C on serine 216 and promotes 14-3-3 protein binding. Cell Growth Differ. 9, 197–208. Pinheiro, E.M., Montell, D.J., 2004. Requirement for Par-6 and Bazooka in Drosophila border cell migration. Development 131, 5243–5251. Prasad, M., Montell, D.J., 2007. Cellular and molecular mechanisms of border cell migration analyzed using time-lapse live-cell imaging. Dev. Cell 12, 997–1005. Rauzi, M., Lenne, P.-F., Lecuit, T., 2010. Planar polarized actomyosin contractile flows control epithelial junction remodelling. Nature 468, 1110–1114. Reig, G., Pulgar, E., Concha, M.L., 2014. Cell migration: from tissue culture to embryos. Development 141, 1999–2013. Reiner, O., Sapir, T., 2014. Mark/Par-1 marking the polarity of migrating neurons. Adv. Exp. Med. Biol. 800, 97–111. Rider, M.H., 2006. The ubiquitin-associated domain of AMPK-related protein kinases allows LKB1-induced phosphorylation and activation. Biochem. J. 394, e7–e9. Ridley, A.J., 2011. Life at the leading edge. Cell 145, 1012–1022. Ridley, A.J., Schwartz, M.A., Burridge, K., Firtel, R.A., Ginsberg, M.H., Borisy, G., Parsons, J.T., Horwitz, A.R., 2003. Cell migration: integrating signals from front to back. Science 302, 1704–1709.

198

Jocelyn A. McDonald

Rodriguez-Boulan, E., Macara, I.G., 2014. Organization and execution of the epithelial polarity programme. Nat. Rev. Mol. Cell Biol. 15, 225–242. Roignot, J., Peng, X., Mostov, K., 2013. Polarity in mammalian epithelial morphogenesis. Cold Spring Harb. Perspect. Biol. 5, a013789. Roversi, G., Pfundt, R., Moroni, R.F., Magnani, I., van Reijmersdal, S., Pollo, B., Straatman, H., Larizza, L., Schoenmakers, E.F.P.M., 2005. Identification of novel genomic markers related to progression to glioblastoma through genomic profiling of 25 primary glioma cell lines. Oncogene 25, 1571–1583. Saadat, I., Higashi, H., Obuse, C., Umeda, M., Murata-Kamiya, N., Saito, Y., Lu, H., Ohnishi, N., Azuma, T., Suzuki, A., Ohno, S., Hatakeyama, M., 2007. Helicobacter pylori CagA targets PAR1/MARK kinase to disrupt epithelial cell polarity. Nature 447, 330–333. Sapir, T., Sapoznik, S., Levy, T., Finkelshtein, D., Shmueli, A., Timm, T., Mandelkow, E.-M., Reiner, O., 2008a. Accurate balance of the polarity kinase MARK2/Par-1 is required for proper cortical neuronal migration. J. Neurosci. 28, 5710–5720. Sapir, T., Shmueli, A., Levy, T., Timm, T., Elbaum, M., Mandelkow, E.-M., Reiner, O., 2008b. Antagonistic effects of doublecortin and MARK2/Par-1 in the developing cerebral cortex. J. Neurosci. 28, 13008–13013. Schaar, B.T., Kinoshita, K., McConnell, S.K., 2004. Doublecortin microtubule affinity is regulated by a balance of kinase and phosphatase activity at the leading edge of migrating neurons. Neuron 41, 203–213. Schmoranzer, J., Fawcett, J.P., Segura, M., Tan, S., Vallee, R.B., Pawson, T., Gundersen, G.G., 2009. Par3 and Dynein associate to regulate local microtubule dynamics and centrosome orientation during migration. Curr. Biol. 19, 1065–1074. Segu, L., Pascaud, A., Costet, P., Darmon, M., Buhot, M.-C., 2008. Impairment of spatial learning and memory in ELKL Motif Kinase1 (EMK1/MARK2) knockout mice. Neurobiol. Aging 29, 231–240. Shelly, M., Poo, M.-M., 2011. Role of LKB1-SAD/MARK pathway in neuronal polarization. Dev. Neurobiol. 71, 508–527. Shin, K., Wang, Q., Margolis, B., 2007. PATJ regulates directional migration of mammalian epithelial cells. EMBO Rep. 8, 158–164. Shulman, J.M., Benton, R., St Johnston, D., 2000. The Drosophila homolog of C. elegans PAR-1 organizes the oocyte cytoskeleton and directs oskar mRNA localization to the posterior pole. Cell 101, 377–388. Shutova, M., Yang, C., Vasiliev, J.M., Svitkina, T., 2012. Functions of nonmuscle myosin II in assembly of the cellular contractile system. PLoS One 7, e40814. Silver, D.L., Montell, D.J., 2001. Paracrine signaling through the JAK/STAT pathway activates invasive behavior of ovarian epithelial cells in Drosophila. Cell 107, 831–841. Small, J.V., Geiger, B., Kaverina, I., Bershadsky, A., 2002. Opinion: how do microtubules guide migrating cells? Nat. Rev. Mol. Cell Biol. 3, 957–964. Stehbens, S., Wittmann, T., 2012. Targeting and transport: how microtubules control focal adhesion dynamics. J. Cell Biol. 198, 481–489. Sun, T.-Q., Lu, B., Feng, J.-J., Reinhard, C., Jan, Y.-N., Fantl, W.J., Williams, L.T., 2001. PAR-1 is a Dishevelled-associated kinase and a positive regulator of Wnt signalling. Nat. Cell Biol. 3, 628–636. Sun, W., Zhang, K., Zhang, X., Lei, W., Xiao, T., Ma, J., Guo, S., Shao, S., Zhang, H., Liu, Y., Yuan, J., Hu, Z., Ma, Y., Feng, X., Hu, S., Zhou, J., Cheng, S., Gao, Y., 2004. Identification of differentially expressed genes in human lung squamous cell carcinoma using suppression subtractive hybridization. Cancer Lett. 212, 83–93. Suzuki, A., Hirata, M., Kamimura, K., Maniwa, R., Yamanaka, T., Mizuno, K., Kishikawa, M., Hirose, H., Amano, Y., Izumi, N., Miwa, Y., Ohno, S., 2004. aPKC acts upstream of PAR-1b in both the establishment and maintenance of mammalian epithelial polarity. Curr. Biol. 14, 1425–1435.

Regulation of Cell Migration by Par-1/MARK

199

Szafranski, P., Goode, S., 2007. Basolateral junctions are sufficient to suppress epithelial invasion during Drosophila oogenesis. Dev. Dyn. 236, 364–373. Tanentzapf, G., Smith, C., McGlade, J., Tepass, U., 2000. Apical, lateral, and basal polarization cues contribute to the development of the follicular epithelium during Drosophila oogenesis. J. Cell Biol. 151, 891–904. Thiery, J.P., Acloque, H., Huang, R.Y.J., Nieto, M.A., 2009. Epithelial-mesenchymal transitions in development and disease. Cell 139, 871–890. Timm, T., Li, X.-Y., Biernat, J., Jiao, J., Mandelkow, E., Vandekerckhove, J., Mandelkow, E.-M., 2003. MARKK, a Ste20-like kinase, activates the polarity-inducing kinase MARK/PAR-1. EMBO J. 22, 5090–5101. Vaccari, T., Rabouille, C., Ephrussi, A., 2005. The Drosophila PAR-1 spacer domain is required for lateral membrane association and for polarization of follicular epithelial cells. Curr. Biol. 15, 255–261. Van de Bor, V., Zimniak, G., Ce´re´zo, D., Schaub, S., Noselli, S., 2011. Asymmetric localisation of cytokine mRNA is essential for JAK/STAT activation during cell invasiveness. Development 138, 1383–1393. Vicente-Manzanares, M., Zareno, J., Whitmore, L., Choi, C.K., Horwitz, A.F., 2007. Regulation of protrusion, adhesion dynamics, and polarity by myosins IIA and IIB in migrating cells. J. Cell Biol. 176, 573–580. Vicente-Manzanares, M., Ma, X., Adelstein, R.S., Horwitz, A.R., 2009. Non-muscle myosin II takes centre stage in cell adhesion and migration. Nat. Rev. Mol. Cell Biol. 10, 778–790. Wallingford, J.B., Habas, R., 2005. The developmental biology of Dishevelled: an enigmatic protein governing cell fate and cell polarity. Development 132, 4421–4436. Wang, Y., Riechmann, V., 2007. The role of the actomyosin cytoskeleton in coordination of tissue growth during Drosophila oogenesis. Curr. Biol. 17, 1349–1355. Wang, X., He, L., Wu, Y.I., Hahn, K.M., Montell, D.J., 2010. Light-mediated activation reveals a key role for Rac in collective guidance of cell movement in vivo. Nat. Cell Biol. 12, 591–597. Watanabe, T., Noritake, J., Kaibuchi, K., 2005. Regulation of microtubules in cell migration. Trends Cell Biol. 15, 76–83. Waterman-Storer, C.M., Salmon, E.D., 1997. Actomyosin-based retrograde flow of microtubules in the lamella of migrating epithelial cells influences microtubule dynamic instability and turnover and is associated with microtubule breakage and treadmilling. J. Cell Biol. 139, 417–434. Wittmann, T., Bokoch, G.M., Waterman-Storer, C.M., 2003. Regulation of leading edge microtubule and actin dynamics downstream of Rac1. J. Cell Biol. 161, 845–851. Xue, B., Krishnamurthy, K., Allred, D.C., Muthuswamy, S.K., 2013. Loss of Par3 promotes breast cancer metastasis by compromising cell-cell cohesion. Nat. Cell Biol. 15, 189–200. Yamahashi, Y., Hatakeyama, M., 2013. PAR1b takes the stage in the morphogenetic and motogenetic activity of Helicobacter pylori CagA oncoprotein. Cell Adh. Migr. 7, 11–18. Yamahashi, Y., Saito, Y., Murata-Kamiya, N., Hatakeyama, M., 2011. Polarity-regulating kinase partitioning-defective 1b (PAR1b) phosphorylates guanine nucleotide exchange factor H1 (GEF-H1) to regulate RhoA-dependent actin cytoskeletal reorganization. J. Biol. Chem. 286, 44576–44584. Yang, N., Inaki, M., Cliffe, A., Rørth, P., 2012. Microtubules and Lis-1/NudE/Dynein regulate invasive cell-on-cell migration in Drosophila. PLoS One 7, e40632. Zhang, Y., Guo, H., Kwan, H., Wang, J.-W., Kosek, J., Lu, B., 2007. PAR-1 kinase phosphorylates Dlg and regulates its postsynaptic targeting at the Drosophila neuromuscular junction. Neuron 53, 201–215.