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Polarized epithelia develop distinct cell surface domains, with the apical membrane acquiring characteristic morphological features such as microvilli. Cell polarization is driven by polarity determinants including the evolutionarily conserved partitioning-defective (PAR) proteins that are separated into distinct cortical domains. PAR protein segregation is thought to be a consequence of asymmetric actomyosin contractions. The mechanism of activation of apically polarized actomyosin contractility is unknown. Here we show that the Cdc42 effector MRCK activates myosin-II at the apical pole to segregate aPKC-Par6 from junctional Par3, defining the apical domain. Apically polarized MRCK-activated actomyosin contractility is reinforced by cooperation with aPKC-Par6 downregulating antagonistic RhoA-driven junctional actomyosin contractility, and drives polarization of cytosolic brush border determinants and apical morphogenesis. MRCK-activated polarized actomyosin contractility is required for apical differentiation and morphogenesis in vertebrate epithelia and Drosophila photoreceptors. Our results identify an apical origin of actomyosin-driven morphogenesis that couples cytoskeletal reorganization to PAR polarity signalling.

Summary Mammalian cell cytoskeletal reorganization for efficient directional movement requires tight coordination of actomyosin and microtubule networks. In this study, we show that LRAP35a potentiates microtubule stabilization by promoting CLASP2/EB1 interaction besides its complex formation with MRCK/MYO18A for retrograde actin flow. The alternate regulation of these two networks by LRAP35a is tightly regulated by a series of phosphorylation events that dictated its specificity. Sequential phosphorylation of LRAP35a by Protein Kinase A (PKA) and Glycogen Synthase Kinase-3β (GSK3β) initiates the association of LRAP35a with CLASP2, while subsequent binding and further phosphorylation by Casein Kinase 1δ (CK1δ) induce their dissociation, which facilitates LRAP35a/MRCK association in driving lamellar actomyosin flow. Importantly, microtubule dynamics is directly moderated by CK1δ activity on CLASP2 to regulate GSK3β phosphorylation of the SxIP motifs that blocks EB1 binding, an event countered by LRAP35a interaction and its competition for CK1δ activity. Overall this study reveals an essential role for LRAP35a in coordinating lamellar contractility and microtubule polarization in cell migration.

Mammalian cell cytoskeletal reorganization for efficient directional movement requires tight coordination of actomyosin and microtubule networks. Here we show that LRAP35a potentiated microtubule stabilization by promoting CLASP2/EB1 interaction besides MRCK/MYO18A complex formation for retrograde actin flow. The alternate regulation of these two networks by LRAP35a was tightly regulated by a series of phosphorylation events that dictated its specificity. Sequential phosphorylation of LRAP35a by PKA and GSK3β initiated the association of LRAP35a with CLASP2, while subsequent binding and further phosphorylation by CK1δ induced their dissociation which would facilitate LRAP35a/MRCK association in driving lamellar actomyosin flow. Importantly, microtubule dynamics was directly moderated by CK1δ activity on CLASP2 to regulate GSK3β phosphorylation of the SxIP motifs that block EB1 binding, an event countered by LRAP35a interaction and its competition for CK1δ activity. This study thus reveals an essential role for LRAP35a in coordinating lamellar contractility and microtubule polarization in cell migration.

Myotonic dystrophy kinase-related Cdc42-binding kinase (MRCK) has been shown to localize to the lamella of mammalian cells through its interaction with an adaptor protein, leucine repeat adaptor protein 35a (LRAP35a), which links it with myosin 18A (MYO18A) for activation of the lamellar actomyosin network essential for cell migration. Here, we report the identification of another adaptor protein LRAP25 that mediates MRCK association with LIM kinase 1 (LIMK1). The lamellipodium-localized LRAP25-MRCK complex is essential for the regulation of local LIMK1 and its downstream F-actin regulatory factor cofilin. Functionally, inhibition of either MRCK or LRAP25 resulted in a marked suppression of LIMK1 activity and down-regulation of cofilin phosphorylation in response to aluminum fluoride induction in B16-F1 cells, which eventually resulted in deregulation of lamellipodial F-actin and reorganization of cytoskeletal structures causing defects in cell polarization and motility. These biochemical and functional characterizations thus underline the functional relevance of the LRAP25-MRCK complex in LIMK1-cofilin signaling and the importance of LRAP adaptors as key determinants of MRCK cellular localization and downstream specificities.

Focal adhesions are protein complexes that link metazoan cells to the extracellular matrix through the integrin family of transmembrane proteins. Integrins recruit many proteins to these complexes, referred to as the "adhesome." We used proximity-dependent biotinylation (BioID) in U2OS osteosarcoma cells to label proteins within 15 to 25 nm of paxillin, a cytoplasmic focal adhesion protein, and kindlin-2, which directly binds β integrins. Using mass spectrometry analysis of the biotinylated proteins, we identified 27 known adhesome proteins and 8 previously unknown components close to paxillin. However, only seven of these proteins interacted directly with paxillin, one of which was the adaptor protein Kank2. The proteins in proximity to β integrin included 15 of the adhesion proteins identified in the paxillin BioID data set. BioID also correctly established kindlin-2 as a cell-cell junction protein. By focusing on this smaller data set, new partners for kindlin-2 were found, namely, the endocytosis-promoting proteins liprin β1 and EFR3A, but, contrary to previous reports, not the filamin-binding protein migfilin. A model adhesome based on both data sets suggests that focal adhesions contain fewer components than previously suspected and that paxillin lies away from the plasma membrane. These data not only illustrate the power of using BioID and stable isotope-labeled mass spectrometry to define macromolecular complexes but also enable the correct identification of therapeutic targets within the adhesome.

Cell movement requires forces generated by non-muscle myosin II (NM II) for coordinated protrusion and retraction. The Cdc42/Rac effector MRCK regulates a specific actomyosin network in the lamella essential for cell protrusion and migration. Together with the Rho effector ROK required for cell rear retraction, they cooperatively regulate cell motility and tumour cell invasion. Despite the increasing importance of ROK inhibitors for both experimental and clinical purposes, there is a lack of specific inhibitors for other related kinases such as MRCK. Here, we report the identification of chelerythrine chloride as a specific MRCK inhibitor. Its ability to block cellular activity of MRCK resulted in the specific loss of NM II-associated MLC phosphorylation in the lamella, and the consequential suppression of cell migration.Structured summary of protein interactionsDMPK phosphorylates MYPT by protein kinase assay (View interaction)PAK alpha phosphorylates MLC by protein kinase assay (View interaction)MRCK alpha phosphorylates MLC by protein kinase assay (View interaction)CRIK phosphorylates MLC by protein kinase assay (View interaction)MRCK beta phosphorylates MLC by protein kinase assay (View interaction)ROK alpha phosphorylates MLC by protein kinase assay (View Interaction 1, 2)MRCK beta phosphorylates MYPT by protein kinase assay (View interaction)MRCK alpha phosphorylates MYPT by protein kinase assay (View interaction)ROK alpha phosphorylates MYPT by protein kinase assay (View interaction)MLCK phosphorylates MLC by protein kinase assay (View interaction)

A novel strategy for the development of molecular aptamer beacon for a signal transduction protein, myotonic dystrophy kinase-related Cdc42-binding kinase (MRCK) was proposed in this work. MRCK is an important downstream effector protein of Cdc42 that phosphorylates proteins involved in organizing actin structures responsible for forming stress fibres, lamellipodia or filopodia. The simple method for MAB design could potentially be applied to other aptamers for modification into a protein probe. The MRCK aptamer was modified into a MAB by adding nucleotides on the 5′ end, which are complementary to the 3′ end of the aptamer so as to destroy the existing structure and change it into a MB form. In the absence of MRCK, the MAB remained a hairpin structure. However, in the presence of MRCK, the equilibrium was shifted towards the formation of the MRCK-aptamer complex, resulting in the preference for the MRCK-binding conformer, where a fluorescence-quenching pair added to the 5′ and 3′ ends signaled any protein-dependent conformation change. The development of MABs for signal transduction proteins will have the potential to replace antibodies for diagnostic assays as well as protein studies in cellular imaging.

The mechanism by which Raf-1 antagonizes Rok-α to promote migration and tumorigenesis is revealed., The activity of Raf-1 and Rok-α kinases is regulated by intramolecular binding of the regulatory region to the kinase domain. Autoinhibition is relieved upon binding to the small guanosine triphosphatases Ras and Rho. Downstream of Ras, Raf-1 promotes migration and tumorigenesis by antagonizing Rok-α, but the underlying mechanism is unknown. In this study, we show that Rok-α inhibition by Raf-1 relies on an intermolecular interaction between the Rok-α kinase domain and the cysteine-rich Raf-1 regulatory domain (Raf-1reg), which is similar to Rok-α's own autoinhibitory region. Thus, Raf-1 mediates Rok-α inhibition in trans, which is a new concept in kinase regulation. This mechanism is physiologically relevant because Raf-1reg is sufficient to rescue all Rok-α–dependent defects of Raf-1–deficient cells. Downstream of Ras and Rho, the Raf-1–Rok-α interaction represents a novel paradigm of pathway cross talk that contributes to tumorigenesis and cell motility.

SummaryActomyosin retrograde flow underlies the contraction essential for cell motility. Retrograde flow in both lamellipodia and lamella is required for membrane protrusion and for force generation by coupling to cell adhesion. We report that the Rac/Cdc42-binding kinase MRCK and myosin II-related MYO18A linked by the adaptor protein LRAP35a form a functional tripartite complex, which is responsible for the assembly of lamellar actomyosin bundles and of a subnuclear actomyosin network. LRAP35a binds independently to MYO18A and MRCK. This binding leads to MRCK activation and its phosphorylation of MYO18A, independently of ROK and MLCK. The MRCK complex moves in concert with the retrograde flow of actomyosin bundles, with MRCK being able to influence other flow components such as MYO2A. The promotion of persistent protrusive activity and inhibition of cell motility by the respective expression of wild-type and dominant-negative mutant components of the MRCK complex show it to be crucial to cell protrusion and migration.

Raf kinases relay signals inducing proliferation, differentiation, and survival. The Raf-1 isoform has been extensively studied as the upstream kinase linking Ras activation to the MEK/ERK module. Recently, however, genetic experiments have shown that Raf-1 plays an essential role in counteracting apoptosis, and that it does so independently of its ability to activate MEK. By conditional gene ablation, we now show that Raf-1 is required for normal wound healing in vivo and for the migration of keratinocytes and fibroblasts in vitro. Raf-1–deficient cells show a symmetric, contracted appearance, characterized by cortical actin bundles and by a disordered vimentin cytoskeleton. These defects are due to the hyperactivity and incorrect localization of the Rho-effector Rok-α to the plasma membrane. Raf-1 physically associates with Rok-α in wild-type (WT) cells, and reintroduction of either WT or kinase-dead Raf-1 in knockout fibroblasts rescues their defects in shape and migration. Thus, Raf-1 plays an essential, kinase-independent function as a spatial regulator of Rho downstream signaling during migration.

We have previously described a partial cDNA sequence encoding a RhoGAP protein, GAP25 that is homologous to the recently reported ArhGAP9 and ArhGAP12. We now describe a related new member ArhGAP15 that shares a number of domain similarities, including a pleckstrin homology (PH) domain, a RhoGAP domain and a novel motif N-terminal to the GAP domain. This novel motif was found to be responsible for nucleotide-independent Rac1 binding. Using swop mutants of Rac/Cdc42, we have established that the binding is through the C-terminal half of Rac1. The GAP domain of ArhGAP15 showed specificity towards Rac1 in vitro. The PH domain is required for ArhGAP15 to localize to cell periphery and over-expression of the full-length ArhGAP15, but not the mutant with a partial deletion of the PH domain, resulted in an increase in actin stress fibers and cell contraction. These morphological effects can be attenuated by the co-expression of dominant negative Rac1N17. HeLa cells expressing ArhGAP15 were also resistant to phorbol myristatate acetate treatment, suggesting that ArhGAP15 is a potential regulator of Rac1.

Rho GTPases play pivotal roles in regulating cell morphology. We previously showed that RhoA acts via ROKalpha to counteract the effects of the classical second messenger cyclic AMP on cell shape changes. Here we show that active Cdc42V12 also competes against the cAMP-induced stellate morphology in SH-EP cells. This Cdc42 effect is not mediated by the RhoA/ ROK pathway but rather the related MRCKalpha, a myotonic dystrophy kinase-related Cdc42-binding kinase. Co-expression of a dominant inhibitory MRCKalpha mutant with Cdc42V12 blocks the ability of the GTPase to counteract cAMP, suggesting that MRCK acts downstream of Cdc42 in this process. Cdc42V12 enhances the phosphorylation of myosin light chain (MLC) at the cell periphery and sustains focal adhesion complexes, while MLC kinase inhibitors destroy focal adhesion complexes and impair the Cdc42V12 protective effect. The data suggest that the maintenance of focal adhesion complexes via the regulation of myosin II activity underlies the ability of Cdc42 to protect against the effect of elevated cAMP.

Rho-binding kinase alpha (ROKalpha) is a serine/threonine kinase with multiple functional domains involved in actomyosin assembly. It has previously been documented that the C terminus part of ROKalpha interacts with the N-terminal kinase domain and thereby regulates its catalytic activity. Here we used antibodies against different domains of ROKalpha and were able to reveal some structural aspects that are essential for the specific functions of ROKalpha. Antibodies against the kinase domain revealed that this part of the protein is highly complex and inaccessible. Further experiments confirmed that this domain could undergo inter- and intramolecular interactions in a complex manner, which regulates the kinase catalytic activity. Other antibodies that raised against the coiled-coil domain, Rho binding domain, and the pleckstrin homology (PH) domain were all effective in recognizing the native proteins in an immunoprecipitation assay. Only the anti-Rho binding domain antibodies could activate the kinase independent of RhoA. The PH antibodies had no apparent effects on the catalytic activity but were effective in blocking actomyosin assembly and cell contractility. Likewise, mutations of the PH domains can abrogate its dominant negative effects on actin morphology. The subsequent disruption of endogenous ROK localization to the actomyosin network by overexpressing the PH domain is supportive of a role of the PH domain of ROK in targeting the kinase to these structures.

Background ERM (ezrin, radixin, and moesin) proteins function as membrane-cytoskeletal linkers, and are known to be localized at filopodia and microvilli-like structures. We have shown that Rho-associated kinase (Rho-kinase)/ROKα/ROCK II phosphorylates moesin at Thr-558 at the lower stream of Rho, and the phosphorylation is crucial to the formation of microvilli-like structures (Oshiro, N., Fukata, Y. & Kaibuchi, K. (1998) Phosphorylation of moesin by Rho-associated kinase (Rho-kinase) plays a crucial role in the formation of microvilli-like structures. J. Biol. Chem. 273, 34663– 34666). However, the role of ERM proteins in the formation of filopodia is less well characterized. Results Here we examined the phosphorylation state of ERM during filopodia formation induced by Cdc42 using the antibody recognizing ERM proteins phosphorylated at COOH (C)-terminal threonine. When NIH 3T3 cells were transfected with constitutively active Cdc42 (Cdc42V12), filopodia formation was induced and phosphorylation of ERM at C-terminal threonine was observed at the tip of filopodia, while the phosphorylation levels of ERM were lower and phosphorylated ERM was distributed throughout the cytoplasm in the control cells. We also showed that Myotonic dystrophy kinase- related Cdc42-binding kinase (MRCK) which has been identified as an effector of Cdc42, phosphorylated moesin at C-terminal threonine in a cell-free system. Coexpression of the dominant negative form of MRCK inhibited both the formation of filopodia and accumulation of C-terminal threonine-phosphorylated ERM proteins at filopodia induced by Cdc42V12. Conclusion The formation of filopodia induced by Cdc42 is accompanied by phosphorylation of ERM proteins, and MRCK is a candidate for the kinase that phosphorylates ERM proteins at filopodia.

Cell motility is a highly coordinated multistep process. Uncovering the mechanism of myosin II (MYO2) activation responsible for the contractility underlying cell protrusion and retraction provides clues on how these complementary activities are coordinated. Several protein kinases have been shown to activate MYO2 by phosphorylating the associated myosin light chain (MLC). Recent work suggests that these MLC kinases are strategically localized to various cellular regions during cell migration in a polarized manner. This localization of the kinases together with their specificity in MLC phosphorylation, their distinct enzymatic properties and the distribution of the myosin isoforms generate the specific contractile activities that separately promote the cell protrusion or retraction essential for cell motility.

The myotonic dystrophy kinase-related Cdc42-binding kinase (MRCKalpha) has been implicated in the morphological activities of Cdc42 in nonneural cells. Both MRCKalpha and the kinase-related Rho-binding kinase (ROKalpha) are involved in nonmuscle myosin light-chain phosphorylation and associated actin cytoskeleton reorganization. We now show that in PC12 cells, overexpression of the kinase domain of MRCKalpha and ROKalpha resulted in retraction of neurites formed on nerve growth factor (NGF) treatment, as observed with RhoA. However, introduction of kinase-dead MRCKalpha did not result in NGF-independent neurite outgrowth as observed with dominant negative kinase-dead ROKalpha or the Rho inhibitor C3. Neurite outgrowth induced by NGF or kinase-dead ROKalpha was inhibited by dominant negative Cdc42(N17), Rac1(N17), and the Src homology 3 domain of c-Crk, indicating the participation of common downstream components. Neurite outgrowth induced by either agent was blocked by kinase-dead MRCKalpha lacking the p21-binding domain or by a minimal C-terminal regulatory region consisting of the cysteine-rich domain/pleckstrin homology domain plus a region with homology to citron. The latter region alone was an effective blocker of NGF-induced outgrowth. These results suggest that although ROKalpha is involved in neurite retraction promoted by RhoA, the related MRCKalpha is conversely involved in neurite outgrowth promoted by Cdc42 and Rac.

Regulation of cytoskeletal dynamics is essential for cell shape change and morphogenesis.Drosophila melanogasterembryos offer a well-defined system for observing alterations in the cytoskeleton during the process of cellularization, a specialized form of cytokinesis. During cellularization, the actomyosin cytoskeleton forms a hexagonal array and drives invagination of the plasma membrane between the nuclei located at the cortex of the syncytial blastoderm. Rho, Rac, and Cdc42 proteins are members of the Rho subfamily of Ras-related G proteins that are involved in the formation and maintenance of the actin cytoskeleton throughout phylogeny and inD. melanogaster.To investigate how Rho subfamily activity affects the cytoskeleton during cellularization stages, embryos were microinjected with C3 exoenzyme fromClostridium botulinumor with wild-type, constitutively active, or dominant negative versions of Rho, Rac, and Cdc42 proteins. C3 exoenzyme ADP-ribosylates and inactivates Rho with high specificity, whereas constitutively active dominant mutations remain in the activated GTP-bound state to activate downstream effectors. Dominant negative mutations likely inhibit endogenous small G protein activity by sequestering exchange factors. Of the 10 agents microinjected, C3 exoenzyme, constitutively active Cdc42, and dominant negative Rho have a specific and indistinguishable effect: the actomyosin cytoskeleton is disrupted, cellularization halts, and embryogenesis arrests. Time-lapse video records of DIC imaged embryos show that nuclei in injected regions move away from the cortex of the embryo, thereby phenocopying injections of cytochalasin or antimyosin. Rhodamine phalloidin staining reveals that the actin-based hexagonal array normally seen during cellularization is disrupted in a dose-dependent fashion. Additionally, DNA stain reveals that nuclei in the microinjected embryos aggregate in regions that correspond to actin disruption. These embryos halt in cellularization and do not proceed to gastrulation. We conclude that Rho activity and Cdc42 regulation are required for cytoskeletal function in actomyosin-driven furrow canal formation and nuclear positioning.

The regulation of morphological changes in eukaryotic cells is a complex process involving major components of the cytoskeleton including actin microfilaments, microtubules, and intermediate filaments (IFs). The putative effector of RhoA, RhoA-binding kinase alpha (ROKalpha), is a serine/threonine kinase that has been implicated in the reorganization of actin filaments and in myosin contractility. Here, we show that ROKalpha also directly affects the structural integrity of IFs. Overexpression of active ROKalpha, like that of RhoA, caused the collapse of filamentous vimentin, a type III IF. A RhoA-binding-deficient, kinase-inactive ROKalpha inhibited the collapse of vimentin IFs induced by RhoA in HeLa cells. In vitro, ROKalpha bound and phosphorylated vimentin at its head-rod domain, thereby inhibiting the assembly of vimentin. ROKalpha colocalized predominantly with the filamentous vimentin network, which remained intact in serum-starved cells. Treatment of cells with vinblastine, a microtubule-disrupting agent, also resulted in filamentous vimentin collapse and concomitant ROKalpha translocation to the cell periphery. ROKalpha translocation did not occur when the vimentin network remained intact in vinblastine-treated cells at 4 degreesC or in the presence of the dominant-negative RhoAN19 mutant. Transient translocation of ROKalpha was also observed in cells subjected to heat shock, which caused the disassembly of the vimentin network. Thus, the translocation of ROKalpha to the cell periphery upon overexpression of RhoAV14 or growth factor treatment is associated with disassembly of vimentin IFs. These results indicate that Rho effectors known to act on microfilaments may be involved in regulating the assembly of IFs. Vimentin when phosphorylated also exhibits reduced affinity for the inactive ROKalpha. The translocation of ROKalpha from IFs to the cell periphery upon action by activated RhoA and ROKalpha suggests that ROKalpha may initiate its own cascade of activation.

AlphaPAK in a constitutively active form can exert morphological effects (E. Manser, H.-Y. Huang, T.-H. Loo, X.-Q. Chen, J.-M. Dong, T. Leung, and L. Lim, Mol. Cell. Biol. 17:1129-1143, 1997) resembling those of Cdc42G12V. PAK family kinases, conserved from yeasts to humans, are directly activated by Cdc42 or Rac1 through interaction with a conserved N-terminal motif (corresponding to residues 71 to 137 in alphaPAK). alphaPAK mutants with substitutions in this motif that resulted in severely reduced Cdc42 binding can be recruited normally to Cdc42G12V-driven focal complexes. Mutation of residues in the C-terminal portion of the motif (residues 101 to 137), though not affecting Cdc42 binding, produced a constitutively active kinase, suggesting this to be a negative regulatory region. Indeed, a 67-residue polypeptide encoding alphaPAK83-149 potently inhibited GTPgammaS-bound Cdc42-mediated kinase activation of both alphaPAK and betaPAK. Coexpression of this PAK inhibitor with Cdc42G12V prevented the formation of peripheral actin microspikes and associated loss of stress fibers normally induced by the p21. Coexpression of PAK inhibitor with Rac1G12V also prevented loss of stress fibers but not ruffling induced by the p21. Coexpression of alphaPAK83-149 completely blocked the phenotypic effects of hyperactive alphaPAKL107F in promoting dissolution of focal adhesions and actin stress fibers. These results, coupled with previous observations with constitutively active PAK, demonstrate that these kinases play an important role downstream of Cdc42 and Rac1 in cytoskeletal reorganization.

The PAK family of kinases are regulated through interaction with the small GTPases Cdc42 and Rac1, but little is known of the signaling components immediately upstream or downstream of these proteins. We have purified and cloned a new class of Rho-p21 guanine nucleotide exchange factor binding tightly through its N-terminal SH3 domain to a conserved proline-rich PAK sequence with a Kd of 24 nM. This PAK-interacting exchange factor (PIX), which is widely expressed and enriched in Cdc42- and Rac1-driven focal complexes, is required for PAK recruitment to these sites. PIX can induce membrane ruffling, with an associated activation of Rac1. Our results suggest a role for PIX in Cdc42-to-Rac1 signaling, involving the PIX/PAK complex.

MRCKα is activated by PDK1 through a PIP3-dependent, kinase-independent mechanism that drives the relocation of both proteins to lamellipodia and regulates lamellipodial retraction and directional migration., Directional cell migration is of paramount importance in both physiological and pathological processes, such as development, wound healing, immune response, and cancer invasion. Here, we report that 3-phosphoinositide-dependent kinase 1 (PDK1) regulates epithelial directional migration and invasion by binding and activating myotonic dystrophy kinase–related CDC42-binding kinase α (MRCKα). We show that the effect of PDK1 on cell migration does not involve its kinase activity but instead relies on its ability to bind membrane phosphatidylinositol (3,4,5)-trisphosphate. Upon epidermal growth factor (EGF) stimulation, PDK1 and MRCKα colocalize at the cell membrane in lamellipodia. We demonstrate that PDK1 positively modulates MRCKα activity and drives its localization within lamellipodia. Likewise, the retraction phase of lamellipodia is controlled by PDK1 through an MRCKα-dependent mechanism. In summary, we discovered a functional pathway involving PDK1-mediated activation of MRCKα, which links EGF signaling to myosin contraction and directional migration.

Whether the Hippo pathway has downstream targets other than YAP and TAZ is unknown. In this report, we have identified angiomotin (Amot) family members as novel substrates of Hippo core kinases. The N-terminal regions of Amot proteins contain a conserved HXRXXS consensus site for LATS1/2-mediated phosphorylation. Phospho-specific antibodies showed that Hippo core kinases could mediate phosphorylation of endogenous as well as exogenous Amot family members. Knockdown of LATS1 and LATS2 endogenously reduced the phosphorylation of Amots detected by the phospho-specific antibodies. Mutation of the serine to alanine within this HXRXXS site in Amot and AmotL2 established that this site was essential for Hippo core kinase-mediated phosphorylation. Wild-type and non-phosphorylated Amot (Amot-S175A) were targeted to actin filaments, whereas phospho-mimic Amot (Amot-S175D) failed to be localized with actin. Overexpression of LATS2 caused dissociation of Amot from actin but not Amot-S175A. Mapping of the actin-binding site of Amot showed that serine 175 of Amot was important for the actin-binding activity. Amot-S175A promoted, whereas Amot and Amot-S175D inhibited, cell proliferation. These results collectively suggest that the Hippo pathway negatively regulates the actin-binding activity of Amot family members through direct phosphorylation.

Tools for studying replication fork dynamics are critical for dissecting the mechanisms of DNA replication, DNA repair, histone deposition, and epigenetic memory. Isolation of protein on nascent DNA (iPOND) is an elegant method for purifying replication fork proteins. Here, we present accelerated native iPOND (aniPOND), a simplification of the iPOND procedure with improved protein yield. Cell membrane lysis and nuclei harvesting are combined in one step to reduce washes and minimize sample loss. A mild nuclei lysis protocol is then used to better preserve DNA-protein complexes. aniPOND is faster than iPOND, avoids formaldehyde cross-linking, and improves protein yield 5- and 20-fold for the CAF1-complex or PCNA respectively. Moreover, using aniPOND, but not iPOND, we could detect the polycomb repressive complex 2 (PRC2) components SUZ12, EZH2, and RBBP4 at replication forks. This faster, higher-yield method will facilitate MS analysis of replication fork complexes.

Changes in cell morphology are essential in the development of a multicellular organism. The regulation of the cytoskeleton by the Rho subfamily of small GTP-binding proteins is an important determinant of cell shape. The Rho subfamily has been shown to participate in a variety of morphogenetic processes during Drosophila melanogaster development. We describe here a Drosophila homolog, DPAK, of the serine/threonine kinase PAK, a protein which is a target of the Rho subfamily proteins Rac and Cdc42. Rac, Cdc42, and PAK have previously been implicated in signaling by c-Jun amino-terminal kinases. DPAK bound to activated (GTP-bound) Drosophila Rac (DRacA) and Drosophila Cdc42. Similarities in the distributions of DPAK, integrin, and phosphotyrosine suggested an association of DPAK with focal adhesions and Cdc42- and Rac-induced focal adhesion-like focal complexes. DPAK was elevated in the leading edge of epidermal cells, whose morphological changes drive dorsal closure of the embryo. We have previously shown that the accumulation of cytoskeletal elements initiating cell shape changes in these cells could be inhibited by expression of a dominant-negative DRacA transgene. We show that leading-edge epidermal cells flanking segment borders, which express particularly large amounts of DPAK, undergo transient losses of cytoskeletal structures during dorsal closure. We propose that DPAK may be regulating the cytoskeleton through its association with focal adhesions and focal complexes and may be participating with DRacA in a c-Jun amino-terminal kinase signaling pathway recently demonstrated to be required for dorsal closure.

A number of "target" proteins for the Rho family of small GTP-binding proteins have now been identified, including the protein kinases ACK and p65PAK (Manser, E., Leung, T., Salihuddin, H., Zhao, Z.-S., and Lim, L. (1994) Nature 367, 40-46). The purified serine/threonine kinase p65PAK has been shown to be directly activated by GTP-Rac1 or GTP-Cdc42. Here we report the cDNA sequence encoding a new brain-enriched PAK isoform beta-PAK, which shares 79% amino acid identity with the previously described alpha-isoform. Their mRNAs are differentially expressed in the brain, with alpha-PAK mRNA being particularly abundant in motor-associated regions. In vitro translation products of the alpha- and beta-PAK cDNAs exhibited relative molecular masses of 68,000 and 65,000, respectively, by SDS-polyacrylamide analysis. A specific beta-PAK peptide sequence was obtained from rat brain-purified p65PAK. Recombinant alpha- and beta-PAKs exhibited an increase in kinase activity mediated by GTP-p21 induced autophosphorylation. Cdc42 was a more potent activator in vitro of alpha-PAK kinase, and the fully activated enzyme is 300 times more active than the unphosphorylated form. Interestingly the down-regulation in the binding of p21s to recombinant beta-PAK and brain p65PAK, which is observed upon kinase activation does not occur with recombinant alpha-PAK.

beta-Chimaerin, a 30-kDa GTPase-activating protein (GAP) for the p21 Ras-related Rac, is expressed specifically in late stage spermatocytes (Leung, T., How, B.-E., Manser, E., and Lim, L. (1993) J. Biol. Chem. 268, 3813-3816). Using antibodies raised against beta-chimaerin, we detected a major 46-kDa RacGAP in the rat cerebellum. With beta-chimaerin cDNA as a probe and using polymerase chain reaction, cDNAs from both human and rat cerebellum were isolated. The human and rat cDNAs encoded sequences containing cysteine-rich and GAP domains identical to those of testis beta-chimaerin. The cDNAs also encoded an additional N-terminal SH2 (Src homology 2) domain, probably derived from the beta-chimaerin gene by alternate splicing. This SH2 domain of the predicted 54-kDa protein was strikingly similar to that of alpha 2-chimaerin, including replacement by glutamic acid of the invariant tryptophan present at the start of other SH2 domains. The SH2 domains of alpha- and beta-chimaerin thus represent a subset of SH2 domains. The cerebellar beta-chimaerin (beta 2-) is expressed mainly in granule cells and exhibits postnatal developmental increases. beta 2-Chimaerin was enriched in particulate/synaptosomal fractions. In the mouse weaver mutant lacking mature granule cells, there is a corresponding decrease in beta 2-chimaerin, which could well serve as a marker of granule cell differentiation.

GTPase-activating proteins (GAPs) modulate the activity of the ras superfamily of proteins by converting active GTP-bound to inactive GDP-bound p21s. Employing a novel GAP overlay assay (Manser, E., Leung, T., Monfries, C., Teo, M., Hall, C., and Lim, L. (1992) J. Biol. Chem. 267, 16025-16028), we demonstrated a diversity of proteins with GAP activities in different tissues. Using a polymerase chain reaction strategy exploiting conserved residues in the GAP domains of n-chimaerin and the product of the breakpoint cluster region gene (BCR), we isolated a human brain 5.3-kilobase cDNA containing a 486-base pair region with complete identity to a previously reported active BCR-related (ABR) gene sequence on human chromosome 17. The brain cDNA encoded a 98-kDa protein (ABR) resembling BCR (68% identity), containing both the oncogene dbl-related domain at the N terminus and the GAP domain at the C terminus; however, it lacks the N-terminal BCR protein kinase domain. The ABR GAP domain expressed as an Escherichia coli fusion protein was active against Rac1 and Cdc42 of the rho subfamily. The ABR mRNA is highly enriched in the brain. ABR probably corresponds to the brain-enriched 100-kDa GAP for Rac and Cdc42Hs previously detected. The relationship of ABR to Miller-Dieker syndrome, a neurological disorder co-mapping to 17p13.3, is discussed.

The p21ras superfamily, involved in diverse processes including cell growth and intracellular trafficking, possesses intrinsic GTPase activity and cycles between GTP-bound active and GDP-bound quiescent states. This intrinsic activity, which results in down-regulation, is accelerated by GTPase activating proteins (GAPs). Other proteins regulating the GDP/GTP cycle include exchange proteins and dissociation inhibitors. The p21s rho, rac, and cdc42Hs constitute a subfamily implicated in cytoskeletal organization. BCR and n-chimaerin are prototypes of a new GAP family for these p21s. To investigate proteins modulating GTP hydrolysis of the three p21s, we developed a novel overlay assay applicable to tissue extracts. Diverse GAPs with different specificities were identified in all rat tissues. Brain contained rac1 GAPs of 45, 50, 85, 100, and 150 kDa. The p50 and p150 GAPs also act on rhoA and cdc42Hs and are ubiquitous, while the p45-GAP, n-chimaerin, is brain- and testis-specific and acts preferentially on rac1; the p100 GAP acts on both rac1 and cdc42Hs and is brain-specific. A new class of p21-interacting proteins was also identified. This diversity, versatility, and tissue specificity of GAPs may be required for fine control of the down-regulation of GTP-bound p21s and the suggested specific downstream effects of individual GAPs, which could involve "cross-talk" between GAPs and p21s.

The human heat-shock protein multigene family comprises several highly conserved proteins with structural and functional properties in common, but which vary in the extent of their inducibility in response to metabolic stress. We have isolated and characterized a novel human HSP70 cDNA, HSP70B′ cDNA, and its corresponding gene sequence. HSP70B′ cDNA hybrid-selected an mRNA encoding a more basic 70 kDa heat-shock protein that both the major stress-inducible HSP70 and constitutively expressed HSC70 heat-shock proteins, which in common with other heat-shock 70 kDa proteins bound ATP. The complete HSP70B′ gene was sequenced and, like the major inducible HSP70 gene, is devoid of introns. The HSP70B′ gene has 77% sequence similarity to the HSP70 gene and 70% similarity to HSC70 cDNA, with greatest sequence divergence towards the 3′-terminus. The HSP70B′ gene represents a functional gene, as indicated by Northern-blot analysis with specific oligonucleotides, hybrid-selected translation with a specific 3′ cDNA sequence and S1 nuclease protection experiments. In contrast with HSP70 mRNA, which is present at low concentrations in HeLa cells and readily induced by heat or CdCl2 treatment in both fibroblasts and HeLa cells, HSP70B′ mRNA was induced only at higher temperature and showed no basal expression. The differences in patterns of induction may be due to the special features of the promoter region of the HSP70B′ gene.

Myosin phosphatase targeting subunit 3 (MYPT3) and transforming growth factor-beta-inhibited membrane-associated protein (TIMAP) are two closely related myosin-binding targeting subunits of protein phosphatase 1 (PP1c) with a characteristic CAAX (where AA indicates aliphatic amino acid) box at the C termini. Here we show that MYPT3 can be a substrate for protein kinase A (PKA). We first mapped the multiple phosphorylation sites within a central conserved motif. Deletion or mutations of this motif resulted in enhancement of the associated PP1c activity, suggesting that phosphorylation of MYPT3 may play an important role in regulating PP1c catalytic activity. However, unlike the other known MYPTs, which upon phosphorylation inhibit PP1c, PKA phosphorylation of MYPT3 resulted in PP1c activation, indicating a different mode of action. There is a direct interaction between the central conserved phosphorylated site motif with the N-terminal ankyrin repeat region; this interaction was significantly reduced with MYPT3 phosphorylation or acidic phosphorylation site mutations, with concomitant alterations in biochemical and morphological consequences. We therefore propose a novel mechanism for the phosphorylation of MYPT3 by PKA and activation of the catalytic activity through direct interaction of a central region of MYPT3 with its N-terminal region.

Neurite outgrowth is influenced by positive and negative signals that include the semaphorins, an important family of axonal outgrowth inhibitors. Here we report that the Rac GTPase activating protein (GAP)alpha2-chimaerin is involved in Semaphorin 3A (Sema 3A) signaling. In dorsal root ganglion neurons, Sema 3A-induced growth cone collapse was inhibited by alpha2-chimaerin mutated to eliminate GAP activity or interaction with phosphotyrosine. Activation of alpha2-chimaerin by phorbol ester caused growth cone collapse. Active alpha2-chimaerin interacts with collapsin response mediator protein-2 (CRMP-2) and cyclin-dependent kinase (Cdk) 5/p35 kinase through its SH2 and GAP domains, respectively. Cdk5 phosphorylates CRMP-2 at serine 522, possibly facilitating phosphorylation of serine 518 and threonine 514 by glycogen synthase kinase 3beta (GSK3beta), a kinase previously implicated in Sema 3A signaling. Phosphorylation of CRMP-2 serine 522 was essential for Sema 3A-induced growth cone collapse, which is dependent on Cdk5 but not Rho kinase activity. alpha2-chimaerin, like CRMP-2, can associate with the Sema 3A receptor. These results indicate that active alpha2-chimaerin Rac GAP, Cdk5/p35, and its substrate CRMP-2, are implicated in the dynamics of growth cone guidance initiated through Sema 3A signaling.

Myotonic dystrophy kinase-related Cdc42 binding kinases (MRCKs) are family members most related to the myotonic dystrophy kinase (DMPK), RhoA-binding kinase (ROK), and citron kinase. Two highly conserved members, MRCKalpha and -beta, have been previously identified and characterized. We now describe a novel isoform, MRCKgamma, which is functionally and structurally related to members of this kinase family. We show these kinases to have marked similarities in their genomic organization, substrate phosphorylation, and catalytic autoinhibition. Unlike MRCKalpha and -beta, which are expressed ubiquitously, MRCKgamma mRNA was only expressed in heart and skeletal muscle. In cultured cells, MRCKgamma showed differential expression with high levels of expression only in certain cell lines. DNA analysis showed that lack of expression is correlated with promoter DNA methylation. We have mapped the methylation sites in the MRCKgamma promoter. Significantly, agents that suppressed DNA methylation caused increases in the expression of the kinase in low-expressing cells, further supporting the notion that promoter DNA methylation plays an important role in the expression of MRCKgamma. Analysis of the MRCKgamma promoter has also revealed two proximal Sp1 sites that are essential for transcriptional activity. We conclude that both promoter DNA methylation and Sp1 binding are important regulators for MRCKgamma expression.

Corneal keratocytes (stromal cells) are activated to fibroblasts and myofibroblasts during wound healing. Myofibroblast transdifferentiation is accompanied by the expression of alpha-smooth muscle actin (alpha-SMA) and the assembly of a robust stress fiber network and larger focal adhesions (FAs). The regulation of the assembly of stress fibers was evaluated in cultured corneal fibroblast and myofibroblast phenotypes. In both cell types, the inhibition of Rho GTPase activity by microinjecting C3 transferase into the cells resulted in the disassembly of stress fibers and FAs. However, the inhibition of the Rho-associated kinases ROKalpha and ROKbeta with their inhibitor, Y27632, or by overexpression of their mutant kinase-dead forms resulted in only a partial loss of the stress fibers and FAs in myofibroblasts but a total loss in fibroblasts. ROK inhibitor-sensitive and -resistant stress fibers in myofibroblasts contained alpha-SMA, nonmuscle myosin II, tropomyosin, and calponin. The ROK inhibition-resistant stress fibers and FAs were lost upon the overexpression of the dominant-negative form of mDia1 (a mammalian homolog of Drosophila diaphanous protein). These findings indicated that while the assembly of stress fibers in fibroblasts critically involves both ROK and mDia1, in myofibroblasts, the assembly of alpha-SMA-containing stress fibers also occurs independently of ROK and involves Rho/mDia1.

Rho family GTPases play a major role in actin cytoskeleton reorganization. Recent studies have shown that the activation of Rho family GTPases also induces collapse of the vimentin intermediate filament (IF) network in fibroblasts. Here, we report that Cdc42V12 induces the reorganization of vimentin IFs in Hela cells, and such reorganization is independent of actin and microtubule status. We analyzed the involvement of three serine/threonine kinase effectors, MRCK, PAK and p70 S6K in the Cdc42-induced vimentin reorganization. Surprisingly, the ROK-related MRCK is not involved in this IF reorganization. We detected phosphorylation of vimentin Ser72, a site phosphorylated by PAK, after Cdc42 activation. PAK inhibition partially blocked Cdc42-induced vimentin IF collapse suggesting the involvement of other effectors. We report that p70 S6 kinase (S6K)1 participates in this IF rearrangement since the inhibitor rapamycin or a dominant inhibitory S6K could reduce the Cdc42V12 or bradykinin-induced vimentin collapse. Further, inhibition of PAK and S6K in combination very effectively prevents Cdc42-induced vimentin IF collapse. Conversely, only in combination active PAK and S6K could induce a vimentin IF rearrangement that mimics the Cdc42 effect. Thus, Cdc42-induced vimentin reorganization involves PAK and, in a novel cytoskeletal role, p70 S6K.

The myotonic dystrophy kinase-related kinases RhoA binding kinase and myotonic dystrophy kinase-related Cdc42 binding kinase (MRCK) are effectors of RhoA and Cdc42, respectively, for actin reorganization. Using substrate screening in various tissues, we uncovered two major substrates, p130 and p85, for MRCKalpha-kinase. p130 is identified as myosin binding subunit p130, whereas p85 is a novel related protein. p85 contains N-terminal ankyrin repeats, an alpha-helical C terminus with leucine repeats, and a centrally located conserved motif with the MRCKalpha-kinase phosphorylation site. Like MBS130, p85 is specifically associated with protein phosphatase 1delta (PP1delta), and this requires the N terminus, including the ankyrin repeats. This association is required for the regulation of both the catalytic activities and the assembly of actin cytoskeleton. The N terminus, in association with PP1delta, is essential for actin depolymerization, whereas the C terminus antagonizes this action. The C-terminal effects consist of two independent events that involved both the conserved phosphorylation inhibitory motif and the alpha-helical leucine repeats. The former was able to interact with PP1delta only in the phosphorylated state and result in inactivation of PP1delta activity. This provides further evidence that phosphorylation of a myosin binding subunit protein by specific kinases confers conformational changes in a highly conserved region that plays an essential role in the regulation of its catalytic subunit activities.

Myotonic dystrophy kinase-related Cdc42-binding kinase (MRCK) is a Cdc42-binding serine/threonine kinase with multiple functional domains. We had previously shown MRCKalpha to be implicated in Cdc42-mediated peripheral actin formation and neurite outgrowth in HeLa and PC12 cells, respectively. Here we demonstrate that native MRCK exists in high-molecular-weight complexes. We further show that the three independent coiled-coil (CC) domains and the N-terminal region preceding the kinase domain are responsible for intermolecular interactions leading to MRCKalpha multimerization. N terminus-mediated dimerization and consequent transautophosphorylation are critical processes regulating MRCKalpha catalytic activities. A region containing the two distal CC domains (CC2 and CC3; residues 658 to 930) was found to interact intramolecularly with the kinase domain and negatively regulates its activity. Its deletion also resulted in an active kinase, confirming a negative autoregulatory role. We provide evidence that the N terminus-mediated dimerization and activation of MRCK and the negative autoregulatory kinase-distal CC interaction are two mutually exclusive events that tightly regulate the catalytic state of the kinase. Disruption of this interaction by a mutant kinase domain resulted in increased kinase activity. MRCK kinase activity was also elevated when cells were treated with phorbol ester, which can interact directly with a cysteine-rich domain next to the distal CC domain. We therefore suggest that binding of phorbol ester to MRCK releases its autoinhibition, allowing N-terminal dimerization and subsequent kinase activation.

Dramatic transient changes resulting in a stellate morphology are induced in many cell types on treatment with agents that enhance intracellular cAMP levels. Thrombin fully protects cells from this inductive effect of cAMP through the thrombin receptor. The protective effect of thrombin was shown to be Rho-dependent. Clostridium botulinum C3 exoenzyme, which inactivates RhoA functions, abolished the ability of thrombin to protect cells from responding to increased cAMP levels. A constitutively activated RhoAV14 mutant protein also prevented cells from responding to cAMP. RhoA can be specifically phosphorylated at Ser-188 by the cAMP-activated protein kinase A (PKA). We demonstrate that RhoAV14A188, which cannot be phosphorylated by PKA in vitro, is more effective than RhoAV14 in preventing cells from responding to cAMP and in inducing actin stress fiber formation. This suggests that PKA phosphorylation of RhoA impairs its biological activity in vivo. ROKalpha, a RhoA-associated serine/threonine kinase can also prevent cells from responding to cAMP with shape changes. Phosphorylation of RhoA by PKA in vitro decreases the binding of RhoA to ROKalpha. These results indicate that RhoA and cAMP have antagonistic roles in regulating cellular morphology and suggest that cAMP-mediated down-regulation of RhoA binding to its effector ROKalpha may be involved in this antagonism.

The Rho GTPases play distinctive roles in cytoskeletal reorganization associated with growth and differentiation. The Cdc42/Rac-binding p21-activated kinase (PAK) and Rho-binding kinase (ROK) act as morphological effectors for these GTPases. We have isolated two related novel brain kinases whose p21-binding domains resemble that of PAK whereas the kinase domains resemble that of myotonic dystrophy kinase-related ROK. These approximately 190-kDa myotonic dystrophy kinase-related Cdc42-binding kinases (MRCKs) preferentially phosphorylate nonmuscle myosin light chain at serine 19, which is known to be crucial for activating actin-myosin contractility. The p21-binding domain binds GTP-Cdc42 but not GDP-Cdc42. The multidomain structure includes a cysteine-rich motif resembling those of protein kinase C and n-chimaerin and a putative pleckstrin homology domain. MRCK alpha and Cdc42V12 colocalize, particularly at the cell periphery in transfected HeLa cells. Microinjection of plasmid encoding MRCK alpha resulted in actin and myosin reorganization. Expression of kinase-dead MRCK alpha blocked Cdc42V12-dependent formation of focal complexes and peripheral microspikes. This was not due to possible sequestration of the p21, as a kinase-dead MRCK alpha mutant defective in Cdc42 binding was an equally effective blocker. Coinjection of MRCK alpha plasmid with Cdc42 plasmid, at concentrations where Cdc42 plasmid by itself elicited no effect, led to the formation of the peripheral structures associated with a Cdc42-induced morphological phenotype. These Cdc42-type effects were not promoted upon coinjection with plasmids of kinase-dead or Cdc42-binding-deficient MRCK alpha mutants. These results suggest that MRCK alpha may act as a downstream effector of Cdc42 in cytoskeletal reorganization.

The family of p21-activated protein kinases (PAKs) appear to be present in all organisms that have Cdc42-like GTPases. In mammalian cells, PAKs have been implicated in the activation of mitogen-activated protein kinase cascades, but there are no reported effects of these kinases on the cytoskeleton. Recently we have shown that a Drosophila PAK is enriched in the leading edge of embryonic epithelial cells undergoing dorsal closure (N. Harden, J. Lee, H.-Y. Loh, Y.-M. Ong, I. Tan, T. Leung, E. Manser, and L. Lim, Mol. Cell. Biol. 16:1896-1908, 1996), where it colocalizes with structures resembling focal complexes. We show here by transfection that in epithelial HeLa cells alpha-PAK is recruited from the cytoplasm to distinct focal complexes by both Cdc42(G12V) and Rac1(G12V), which themselves colocalize to these sites. By deletion analysis, the N terminus of PAK is shown to contain targeting sequences for focal adhesions which indicate that these complexes are the site of kinase function in vivo. Cdc42 and Rac1 cause alpha-PAK autophosphorylation and kinase activation. Mapping alpha-PAK autophosphorylation sites has allowed generation of a constitutively active kinase mutant. By fusing regions of Cdc42 to the C terminus of PAK, activated chimeras were also obtained. Plasmids encoding these different constitutively active alpha-PAKs caused loss of stress fibers when introduced into both HeLa cells and fibroblasts, which was similar to the effect of introducing Cdc42(G12V) or Rac1(G12V). Significantly dramatic losses of focal adhesions were also observed. These combined effects resulted in retraction of the cell periphery after plasmid microinjection. These data support our previous suggestions of a role for PAK downstream of both Cdc42 and Rac1 and indicate that PAK functions include the dissolution of stress fibers and reorganization of focal complexes.

The oncogenic Ras p21 GTPases regulate phosphorylation pathways that underlie a wealth of activities, including growth and differentiation, in organisms ranging from yeast to human. In metazoa, growth factors trigger conversion of Ras from an inactive GDP-bound form to an active GTP-bound form. This activation of Ras leads to activation of Raf. Raf is one of the initial kinases in the cytoplasmic mitogen-activated protein kinase (MAPK) cascade, involving extracellular-signal-regulated kinases (ERK), which culminates in nuclear transcription. The Ras-related subfamily of Rho p21s, including Rho, Rac and Cdc42 are similarly active in their GTP-bound forms. These p21s mediate growth-factor-induced morphological changes involving actin-based cellular structures. For example, in mammalian fibroblasts, Rho mediates the formation of cytoskeletal stress fibres induced by lysophosphatidic acid, while Rac mediates the formation of membrane ruffles induced by platelet-derived growth factor, and Cdc42 mediates the formation of peripheral filopodia by bradykinin. In some cases, factor-induced Rac activation results in Rho activation, and factor-induced Cdc42 activation leads to Rac activation, as determined by specific morphological changes. Although separate Cdc42/Rac and Rac/Rho hierarchies exist, these might not extend into a linear form (i.e. Cdc42--Rac--Rho) since Cdc42 and Rho activities may be competitive or even antagonistic. Thus Cdc42-mediated formation of filopodia is accompanied by loss of stress fibres (whose formation is mediated by Rho). Recently, mammalian kinases that bind to the GTP-bound forms of Rho p21s have been isolated. These kinases include the p21-activated serine/threonine kinase (PAK), which is stimulated by binding to Cdc42 and Rac, and the Rho-binding serine/threonine kinase (ROK), which is not as strongly stimulated by binding. These kinases act as effectors for their p21 partners since they can directly affect the reorganization of the relevant actin-containing structures. ROK promotes the formation of Rho-induced actin-containing stress fibres and focal-adhesion complexes, to which the ends of the stress fibres attach. PAK stimulates the disassembly of stress fibres, which has been shown to accompany formation of Cdc42-induced peripheral-actin-containing structures, including filopodia, which with Rac-induced membrane ruffles play a role in cell movement. PAK also fosters loss of focal-adhesion complexes. Thus, there is cooperation between different Rho p21s as well as antagonism, with their associated kinases having a role in the integration of the reorganization of the actin cytoskeleton. The similarity of PAK to the Saccharomyces cerevisiae kinase Ste20p, which initiates the yeast mating/pheromone MAPK cascade, led to experiments showing that Cdc42 regulates Ste20p in this MAPK pathway. This similarity has also led to the demonstration that mammalian Cdc42 and Rac can signal to the nucleus through MAPK pathways. However, c-Jun N-terminal kinase (JNK, stress-activated protein kinase) rather than ERK, is involved. PAK have been implicated in the JNK pathway, but their exact roles are uncertain. Thus members of the Rho subfamily, and kinases that bind to these p21s are intimately involved in immediate morphological processes as well as long-term transcriptional events.

The GTPase RhoA has been implicated in various cellular activities, including the formation of stress fibers, motility, and cytokinesis. We recently reported on a p150 serine/threonine kinase (termed ROK alpha) binding RhoA only in its active GTP-bound state and on its cDNA; introduction of RhoA into HeLa cells resulted in translocation of the cytoplasmic kinase to plasma membranes, consistent with ROK alpha being a target for RhoA (T. Leung, E. Manser, L. Tan, and L. Lim, J. Biol. Chem. 256:29051-29054, 1995). Reanalysis of the cDNA revealed that ROK alpha contains an additional N-terminal region. We also isolated another cDNA which encoded a protein (ROK beta) with 90% identity to ROK alpha in the kinase domain. Both ROK alpha and ROK beta, which had a molecular mass of 160 kDa, contained a highly conserved cysteine/histidine-rich domain located within a putative pleckstrin homology domain. The kinases bound RhoA, RhoB, and RhoC but not Rac1 and Cdc42. The Rho-binding domain comprises about 30 amino acids. Mutations within this domain caused partial or complete loss of Rho binding. The morphological effects of ROK alpha were investigated by microinjecting HeLa cells with DNA constructs encoding various forms of ROK alpha. Full-length ROK alpha promoted formation of stress fibers and focal adhesion complexes, consistent with its being an effector of RhoA. ROK alpha truncated at the C terminus promoted this formation and also extensive condensation of actin microfilaments and nuclear disruption. The proteins exhibited protein kinase activity which was required for stress fiber formation; the kinase-dead ROK alpha K112A and N-terminally truncated mutants showed no such promotion. The latter mutant instead induced disassembly of stress fibers and focal adhesion complexes, accompanied by cell spreading. These effects were mediated by the C-terminal region containing Rho-binding, cysteine/histidine-rich, and pleckstrin homology domains. Thus, the multidomained ROK alpha appears to be involved in reorganization of the cytoskeleton, with the N and C termini acting as positive and negative regulators, respectively, of the kinase domain whose activity is crucial for formation of stress fibers and focal adhesion complexes.

We previously reported the cloning of a serine/threonine kinase, PAK (for p21 (Cdc42/Rac)-activated kinase), which binds to the Ras-related GTPases Cdc42Hs and Rac1 (Manser, E., Leung, T., Salihuddin, H., Zhao, Z-s., and Lim, L. (1994) Nature 367, 40-46). These p21 proteins together with RhoA comprise the Rho subfamily of proteins that are involved in morphological events. We now report the isolation of a rat cDNA encoding a 150-kDa protein, which specifically binds RhoA in its GTP form and contains an N-terminal serine/threonine kinase domain highly related to the human myotonic dystrophy kinase and a cysteine-rich domain toward the C terminus. The RhoA binding domain is unrelated to other p21 binding domains. Antibody raised against the kinase domain of the predicted protein, termed ROK alpha (for ROK alpha, RhoA-binding kinase), recognized a ubiquitous 150-kDa protein. The brain p150 purified by affinity chromatography with RhoA exhibited serine/threonine kinase activity. In cultured cells, immunoreactive p150 was recruited to membranes upon transfection with dominant positive RhoAV14 mutant and was localized with actin microfilaments at the cell periphery. These results are consistent with a role for the kinase ROK alpha as an effector for RhoA.

Pheromone signalling in Saccharomyces cerevisiae is mediated by the STE4-STE18 G-protein beta gamma subunits. A possible target for the subunits is Ste20p, whose structural homolog, the serine/threonine kinase PAK, is activated by GTP-binding p21s Cdc42 and Rac1. The putative Cdc42p-binding domain of Ste20p, expressed as a fusion protein, binds human and yeast GTP-binding Cdc42p. Cdc42p is required for alpha-factor-induced activation of FUS1.cdc24ts strains defective for Cdc42p GDP/GTP exchange show no pheromone induction at restrictive temperatures but are partially rescued by overexpression of Cdc42p, which is potentiated by Cdc42p12V mutants. Epistatic analysis indicates that CDC24 and CDC42 lie between STE4 and STE20 in the pathway. The two-hybrid system revealed that Ste4p interacts with Cdc24p. We propose that Cdc42p plays a pivotal role both in polarization of the cytoskeleton and in pheromone signalling.

Diverse GTPase-activating proteins (GAPs) for the p21rho subfamily were detected by a novel overlay assay (Manser, E., Leung, T., Monfries, C., Teo, M., Hall, C., and Lim, L. (1992) J. Biol. Chem. 267, 16025-16028), with some GAPs being tissue-specific. Using a PCR strategy exploiting conserved regions common to rho/rac-GAPs, we have isolated a rat testis cDNA encoding a 34-kDa rac-GAP termed beta-chimaerin, as it was highly related to n-chimaerin, containing both a GAP domain (77% identity) and the phorbol ester-binding region (93% identity). beta-Chimaerin mRNA is expressed exclusively in the testis at the onset of sexual maturation. In situ hybridization and cell fractionation analyses show beta-chimaerin mRNA expression to be stage-specific, paralleling acrosomal assembly at the late stage of spermatogenesis. A corresponding testis-specific 30-kDa rac-GAP was detected. The testis-specific and stage-dependent expression of this new member of the chimaerin family offer an alternative model system for investigating the functional role of this class of p21 GAPs, particularly in relation to cytoskeletal reorganization.