Formin DAAM1 Organizes Actin Filaments in the Cytoplasmic Nodal Actin Network.

A nodal cytoplasmic actin network underlies actin cytoplasm cohesion in the absence of stress fibers. We previously described such a network that forms upon Latrunculin A (LatA) treatment, in which formin DAAM1 was localized at these nodes. Knock down of DAAM1 reduced the mobility of actin nodes but the nodes remained. Here we have investigated DAAM1 containing nodes after LatA washout. DAAM1 was found to be distributed between the cytoplasm and the plasma membrane. The membrane binding likely occurs through an interaction with lipid rafts, but is not required for F-actin assembly. Interesting the forced interaction of DAAM1 with plasma membrane through a rapamycin-dependent linkage, enhanced F-actin assembly at the cell membrane (compared to the cytoplasm) after the LatA washout. However, immediately after addition of both rapamycin and LatA, the cytoplasmic actin nodes formed transiently, before DAAM1 moved to the membrane. This was consistent with the idea that DAAM1 was initially anchored to cytoplasmic actin nodes. Further, photoactivatable tracking of DAAM1 showed DAAM1 was immobilized at these actin nodes. Thus, we suggest that DAAM1 organizes actin filaments into a nodal complex, and such nodal complexes seed actin network recovery after actin depolymerization.

Proximity biotinylation provides insight into the molecular composition of focal adhesions at the nanometer scale.

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 ß 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 ß 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 ß1 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.

PAK5 is auto-activated by a central domain that promotes kinase oligomerization.

PAKs (p21 activated kinases) are an important class of Rho effectors. These contain a Cdc42-Rac1 interaction and binding (CRIB) domain and a flanking auto-inhibitory domain (AID) which binds the C-terminal catalytic domain. The group II kinases PAK4 and PAK5 are considered significant therapeutic targets in cancer. Among human cancer cell lines we tested, PAK5 protein levels are much lower than those of PAK4, even in NCI-H446 which has the highest PAK5 mRNA expression. Although these two kinases are evolutionarily and structurally related, it has never been established why PAK4 is inactive whereas PAK5 has high basal activity. The AID of PAK5 is functionally indistinguishable from that of PAK4, pointing to other regions being responsible for higher activity of PAK5. Gel filtration indicates PAK4 is a monomer but PAK5 is dimeric. The central region of PAK5 (residues 109-420) is shown here to promote self-association, and an elevated activity, but has no effect on activation loop Ser(602) phosphorylation. These residues allow PAK5 to form characteristic puncta in cells, and removing sequences involved in oligomerization suppresses kinase activity. Our model suggests PAK5 self-association interferes with AID binding to the catalytic domain, thus maintaining its high activity. Further, our model explains the observation that PAK5 (1-180) inhibits PAK5 in vitro.

An in cellulo-derived structure of PAK4 in complex with its inhibitor Inka1.

PAK4 is a metazoan-specific kinase acting downstream of Cdc42. Here we describe the structure of human PAK4 in complex with Inka1, a potent endogenous kinase inhibitor. Using single mammalian cells containing crystals 50 µm in length, we have determined the in cellulo crystal structure at 2.95 Å resolution, which reveals the details of how the PAK4 catalytic domain binds cellular ATP and the Inka1 inhibitor. The crystal lattice consists only of PAK4-PAK4 contacts, which form a hexagonal array with channels of 80 Å in diameter that run the length of the crystal. The crystal accommodates a variety of other proteins when fused to the kinase inhibitor. Inka1-GFP was used to monitor the process crystal formation in living cells. Similar derivatives of Inka1 will allow us to study the effects of PAK4 inhibition in cells and model organisms, to allow better validation of therapeutic agents targeting PAK4.

The Cdc42 Effector Kinase PAK4 Localizes to Cell-Cell Junctions and Contributes to Establishing Cell Polarity.

The serine/threonine kinase PAK4 is a Cdc42 effector whose role is not well understood; overexpression of PAK4 has been associated with some cancers, and there are reports that correlate kinase level with increased cell migration in vitro. Here we report that PAK4 is primarily associated with cell-cell junctions in all the cell lines we tested, and fails to accumulate at focal adhesions or at the leading edge of migrating cells. In U2OS osteosarcoma and MCF-7 breast cancer cell lines, PAK4 depletion did not affect collective cell migration, but affected cell polarization. By contrast, Cdc42 depletion (as reported by many studies) caused a strong defect in junctional assembly in multiple cells lines. We also report that the depletion of PAK4 protein or treatment of cells with the PAK4 inhibitor PF-3758309 can lead to defects in centrosome reorientation (polarization) after cell monolayer wounding. These experiments are consistent with PAK4 forming part of a conserved cell-cell junctional polarity Cdc42 complex. We also confirm ß-catenin as a target for PAK4 in these cells. Treatment of cells with PF-3758309 caused inhibition of ß-catenin Ser-675 phosphorylation, which is located predominantly at cell-cell junctions.

Myotonic dystrophy kinase-related Cdc42-binding kinases (MRCK), the ROCK-like effectors of Cdc42 and Rac1.

Cdc42 is a member of the Rho GTPase protein family that plays key roles in local F-actin organization through a number of kinase and non-kinase effector proteins. The myotonic dystrophy kinase-related Cdc42-binding kinases (MRCKs), and the RhoA binding coiled-coil containing kinases (ROCKs) are widely expressed members of the Dystrophia myotonica protein kinase (DMPK) family. The MRCK proteins are ∼190 kDa multi-domain proteins expressed in all cells and coordinate certain acto-myosin networks. Notably MRCK is a key regulator of myosin18A and myosin IIA/B, and through phosphorylation of their common regulatory light chains (MYL9 or MLC2) to promote actin stress fiber contractility. The MRCK kinases are regulated by Cdc42, which is required for cell polarity and directional migration; MRCK links to the acto-myosin complex through interaction with a coiled-coil containing adaptor proteins LRAP35a/b. The biological activities of MRCK in model organisms such as worms and flies confirm it as a myosin II activator. In mammalian cell culture MRCK can be critical for cancer cell migration and neurite outgrowth. We review the current literatures regarding MRCK and highlight the similarities and differences between MRCK and ROCK kinases.

Arg kinase-binding protein 2 (ArgBP2) interaction with α-actinin and actin stress fibers inhibits cell migration.

Cell migration requires dynamic remodeling of the actomyosin network. We report here that an adapter protein, ArgBP2, is a component of α-actinin containing stress fibers and inhibits migration. ArgBP2 is undetectable in many commonly studied cancer-derived cell lines. COS-7 and HeLa cells express ArgBP2 (by Western analysis), but expression was detectable only in approximately half the cells by immunofluorescence. Short term clonal analysis demonstrated 0.2-0.3% of cells switch ArgBP2 expression (on or off) per cell division. ArgBP2 can have a fundamental impact on the actomyosin network: ArgBP2 positive COS-7 cells, for example, are clearly distinguishable by their denser actomyosin (stress fiber) network. ArgBP2γ binding to α-actinin appears to underlie its ability to localize to stress fibers and decrease cell migration. We map a small α-actinin binding region in ArgBP2 (residues 192-228) that is essential for these effects. Protein kinase A phosphorylation of ArgBP2γ at neighboring Ser-259 and consequent 14-3-3 binding blocks its interaction with α-actinin. ArgBP2 is known to be down-regulated in some aggressively metastatic cancers. Our work provides a biochemical explanation for the anti-migratory effect of ArgBP2.

NMR binding and crystal structure reveal that intrinsically-unstructured regulatory domain auto-inhibits PAK4 by a mechanism different from that of PAK1.

Six human PAK members are classified into groups I (PAKs 1-3) and II (PAK4-6). Previously, only group I PAKs were thought to be auto-inhibited but very recently PAK4, the prototype of group II PAKs, has also been shown to be auto-inhibited by its N-terminal regulatory domain. However, the complete auto-inhibitory domain (AID) sequence remains undefined and the mechanism underlying its auto-inhibition is largely elusive. Here, the N-terminal regulatory domain of PAK4 sufficient for auto-inhibiting and binding Cdc42/Rac was characterized to be intrinsically unstructured, but nevertheless we identified the entire AID sequence by NMR. Strikingly, an AID peptide was derived by deleting the binding-unnecessary residues, which has a Kd of 320 nM to the PAK4 catalytic domain. Consequently, the PAK4 crystal structure complexed with the entire AID has been determined, which reveals that the complete kinase cleft is occupied by 20 AID residuescomposed of an N-terminal α-helix and a previously-identified pseudosubstrate motif, thus achieving auto-inhibition. Our study reveals that PAK4 is auto-inhibited by a novel mechanism which is completely different from that for PAK1, thus bearing critical implications for design of inhibitors specific for group II PAKs.

The PAK system links Rho GTPase signaling to thrombin-mediated platelet activation.

Regulation of the platelet actin cytoskeleton by the Rho family of small GTPases is essential for the proper maintenance of hemostasis. However, little is known about how intracellular platelet activation from Rho GTPase family members, including Rac, Cdc42, and Rho, translate into changes in platelet actin structures. To better understand how Rho family GTPases coordinate platelet activation, we identified platelet proteins associated with Rac1, a Rho GTPase family member, and actin regulatory protein essential for platelet hemostatic function. Mass spectrometry analysis revealed that upon platelet activation with thrombin, Rac1 associates with a set of effectors of the p21-activated kinases (PAKs), including GIT1, ßPIX, and guanine nucleotide exchange factor GEFH1. Platelet activation by thrombin triggered the PAK-dependent phosphorylation of GIT1, GEFH1, and other PAK effectors, including LIMK1 and Merlin. PAK was also required for the thrombin-mediated activation of the MEK/ERK pathway, Akt, calcium signaling, and phosphatidylserine (PS) exposure. Inhibition of PAK signaling prevented thrombin-induced platelet aggregation and blocked platelet focal adhesion and lamellipodia formation in response to thrombin. Together, these results demonstrate that the PAK signaling system is a key orchestrator of platelet actin dynamics, linking Rho GTPase activation downstream of thrombin stimulation to PAK effector function, MAP kinase activation, calcium signaling, and PS exposure in platelets.

PAK family kinases: Physiological roles and regulation.

The p21-activated kinases (PAKs) are a family of Ser/Thr protein kinases that are represented by six genes in humans (PAK 1-6), and are found in all eukaryotes sequenced to date. Genetic and knockdown experiments in frogs, fish and mice indicate group I PAKs are widely expressed, required for multiple tissue development, and particularly important for immune and nervous system function in the adult. The group II PAKs (human PAKs 4-6) are more enigmatic, but their restriction to metazoans and presence at cell-cell junctions suggests these kinases emerged to regulate junctional signaling. Studies of protozoa and fungal PAKs show that they regulate cell shape and polarity through phosphorylation of multiple cytoskeletal proteins, including microtubule binding proteins, myosins and septins. This chapter discusses what we know about the regulation of PAKs and their physiological role in different model organisms, based primarily on gene knockout studies.

The PAKs come of age: Celebrating 18 years of discovery.

Protein kinases are versatile signaling molecules that are involved in the regulation most physiological responses. The p21-activated kinases (PAKs) can be activated directly by the small GTPases Rac and Cdc42 and are among the best characterized downstream effectors of these Rho proteins. The structure, substrate specificity and functional role of PAKS are evolutionarily conserved from protozoa to mammals. Vertebrate PAKs are particularly important for cytoskeletal remodeling and focal adhesion assembly, thereby contributing to dynamic processes such as cell migration and synaptic plasticity. This issue of Cellular Logistics focuses on the PAK family of kinases, with ten reviews written by researchers currently working in the field. Here in this introductory overview we highlight some of the most interesting recent discoveries regarding PAK biochemistry and biology. The reviews in this issue cover a range of topics including the atomic structures of PAK1 and PAK4, their role in animals as assessed by knockout studies, and how PAKs are likely to contribute to cancer and neurodegenerative diseases. The promise remains that PAK inhibitors will emerge that validate current pre-clinical studies suggesting that blocking PAK activity will positively contribute to human health.

The GTPase-deficient Rnd proteins are stabilized by their effectors.

Rnd proteins are Rho family GTP-binding proteins with cellular functions that antagonize RhoA signaling. We recently described a new Rnd3 effector Syx, also named PLEKHG5, that interacts with Rnds via a Raf1-like "Ras-binding domain." Syx is a multidomain RhoGEF that participates in early zebrafish development. Here we demonstrated that Rnd1, Rnd2, and Rnd3 stability is acutely dependent on interaction with their effectors such as Syx or p190 RhoGAP. Although Rnd3 turnover is blocked by treatment of cells with MG132, we provide evidence that such turnover is mediated indirectly by effects on the Rnd3 effectors, rather than on Rnd3 itself, which is not significantly ubiquitinated. The minimal regions of Syx and p190 RhoGAP that bind Rnd3 are not sequence-related but have similar effects. We have identified features that allow for Rnd3 turnover including a conserved Lys-45 close to the switch I region and the C-terminal membrane-binding domain of Rnd3, which cannot be substituted by the equivalent Cdc42 CAAX sequence. By contrast, an effector binding-defective mutant of Rnd3 when overexpressed undergoes turnover at normal rates. Interestingly the activity of the RhoA-regulated kinase ROCK stimulates Rnd3 turnover. This study suggests that Rnd proteins are regulated through feedback mechanisms in cells where the level of effectors and RhoA activity influence the stability of Rnd proteins. This effector feedback behavior is analogous to the ability of ACK1 and PAK1 to prolong the lifetime of the active GTP-bound state of Cdc42 and Rac1.

Group I and II mammalian PAKs have different modes of activation by Cdc42.

p21-activated kinases (PAKs) are Cdc42 effectors found in metazoans, fungi and protozoa. They are subdivided into PAK1-like (group I) or PAK4-like (group II) kinases. Human PAK4 is widely expressed and its regulatory mechanism is unknown. We show that PAK4 is strongly inhibited by a newly identified auto-inhibitory domain (AID) formed by amino acids 20 to 68, which is evolutionarily related to that of other PAKs. In contrast to group I kinases, PAK4 is constitutively phosphorylated on Ser 474 in the activation loop, but held in an inactive state until Cdc42 binding. Thus, group II PAKs are regulated through conformational changes in the AID rather than A-loop phosphorylation.

PAKs in human disease.

The p21-activated kinases (PAKs) are one of the first direct kinase targets of Ras-related small GTPases to be discovered and have emerged as central players in growth factor signaling networks that regulate morphogenetic processes. In some situations, PAKs control cell proliferation, but their wider role involves establishing cell polarity and promoting cellular plasticity via changes in the actin cytoskeleton. PAKs have been shown to impact on three important areas of human health, namely, cancer, brain function, and virus infection. We review the mechanisms and targets of PAKs in these contexts and provide an overview of the ways in which inhibitors might act to arrest tumor growth, combat virus infection, and promote cell apoptosis. Although PAKs are most abundant in the brain, there are few details of how they might be operating in this context. The advent of new and more selective PAK inhibitors promises new avenues of treatment and allows us to probe in greater detail the importance of PAK biology.

Collapsin response mediator proteins (CRMPs) are a new class of microtubule-associated protein (MAP) that selectively interacts with assembled microtubules via a taxol-sensitive binding interaction.

Collapsin response mediator proteins are ubiquitously expressed from multiple genes (CRMPs 1-5) and play important roles in dividing cells and during semaphorin 3A (Sema3A) signaling. Nonetheless, their mode of action remains opaque. Here we carried out in vivo and in vitro assays that demonstrate that CRMPs are a new class of microtubule-associated protein (MAP). In experiments with CRMP1 or CRMP2 and their derivatives, only the C-terminal region (residues 490-572) mediated microtubule binding. The in vivo microtubule association of CRMPs was abolished by taxol or epothilone B, which is highly unusual. CRMP2-depleted cells exhibited destabilized anaphase astral microtubules and altered spindle position. In a cell-based assay, all CRMPs stabilized interphase microtubules against nocodazole-mediated depolymerization, with CRMP1 being the most potent. Remarkably, a 82-residue C-terminal region of CRMP1 or CRMP2, unrelated to other microtubule binding motifs, is sufficient to stabilize microtubules. In cells, we demonstrate that glycogen synthase kinase-3ß (GSK3ß) inhibition potentiates this activity. Thus, CRMPs are a new class of MAP that binds through a unique motif, but in common with others such as Tau, is antagonized by GSK3ß. This regulation is consistent with such kinases being critical for the Sema3A (collapsin) pathway. These findings have implications for cancer and neurodegeneration.

The Cdc42-associated kinase ACK1 is not autoinhibited but requires Src for activation.

The non-RTK (receptor tyrosine kinase) ACK1 [activated Cdc42 (cell division cycle 42)-associated kinase 1] binds a number of RTKs and is associated with their endocytosis and turnover. Its mode of activation is not well established, but models have suggested that this is an autoinhibited kinase. Point mutations in its SH3 (Src homology 3)- or EGF (epidermal growth factor)-binding domains have been reported to activate ACK1, but we find neither of the corresponding W424K or F820A mutations do so. Indeed, deletion of the various ACK1 domains C-terminal to the catalytic domain are not associated with increased activity. A previous report identified only one major tyrosine phosphorylated protein of 60 kDa co-purified with ACK1. In a screen for new SH3 partners for ACK1 we found multiple Src family kinases; of these c-Src itself binds best. The SH2 and SH3 domains of Src interact with ACK1 Tyr518 and residues 623-652 respectively. Src targets the ACK1 activation loop Tyr284, a poor autophosphorylation site. We propose that ACK1 fails to undergo significant autophosphorylation on Tyr284 in vivo because it is basophilic (whereas Src is acidophilic). Subsequent ACK1 activation downstream of receptors such as EGFR (EGF receptor) (and Src) promotes turnover of ACK1 in vivo, which is blocked by Src inhibitors, and is compromised in the Src-deficient SYF cell line. The results of the present study can explain why ACK1 is responsive to so many external stimuli including RTKs and integrin ligation, since Src kinases are commonly recruited by multiple receptor systems.

A robust protocol to map binding sites of the 14-3-3 interactome: Cdc25C requires phosphorylation of both S216 and S263 to bind 14-3-3.

Modern proteomic techniques have identified hundreds of proteins that bind 14-3-3s, the most widespread eukaryotic phosphoserine/threonine sensors, but accurate prediction of the target phospho-sites is difficult. Here we describe a systematic approach using synthetic peptides that tests large numbers of potential binding sites in parallel for human 14-3-3. By profiling the sequence requirements for three diverse 14-3-3 binding sites (from IRS-1, IRSp53 and GIT2), we have generated enhanced bioinformatics tools to score sites and allow more tractable testing by co-immunoprecipitation. This approach has allowed us to identify two additional sites other than Ser216 in the widely studied cell division cycle (Cdc) protein 25C, whose function depends on 14-3-3 binding. These Ser247 and Ser263 sites in human Cdc25C, which were not predicted by the existing Scansite search, are conserved across species and flank the nuclear localization region. Furthermore, we found strong interactions between 14-3-3 and peptides with the sequence Rxx[S/T]xR typical for PKC sites, and which is as abundant as the canonical Rxx[S/T]xP motif in the proteome. Two such sites are required for 14-3-3 binding in the polarity protein Numb. A recent survey of >200 reported sites identified only a handful containing this motif, suggesting that it is currently under-appreciated as a candidate binding site. This approach allows one to rapidly map 14-3-3 binding sites and has revealed alternate motifs.

Do PAKs make good drug targets?

p21-activated kinases (PAKs) act downstream of Rho-family GTPase and are linked to steps in both cancer initiation and progression. There are six mammalian PAK isoforms that are divided into two groups, and for different reasons both groups are attractive targets for cancer therapy. We describe the background and recent development of a PAK inhibitor, PF-3758309, which exhibits relatively good selectivity and high potency for PAKs. Experiments using PF-3758309 confirm that inhibiting PAK is a beneficial strategy to combat some tumors, and this activity is likely related to modulation of both cell proliferation and survival. The genetic loss of NF2 (neurofibromatosis type 2) leading to increased cell proliferation through a Ras-Rac-PAK pathway may represent a good test system to analyze this new PAK inhibitor.

DAAM1 is a formin required for centrosome re-orientation during cell migration.

BACKGROUND: Disheveled-associated activator of morphogenesis 1 (DAAM1) is a formin acting downstream of Wnt signaling that is important for planar cell polarity. It has been shown to promote proper cell polarization during embryonic development in both Xenopus and Drosophila. Importantly, DAAM1 binds to Disheveled (Dvl) and thus functions downstream of the Frizzled receptors. Little is known of how DAAM1 is localized and functions in mammalian cells. We investigate here how DAAM1 affects migration and polarization of cultured cells and conclude that it plays a key role in centrosome polarity. METHODOLOGY/PRINCIPAL FINDINGS: Using a specific antibody to DAAM1, we find that the protein localizes to the acto-myosin system and co-localizes with ventral myosin IIB-containing actin stress fibers. These fibers are particularly evident in the sub-nuclear region. An N-terminal region of DAAM1 is responsible for this targeting and the DAAM1(1-440) protein can interact with myosin IIB fibers independently of either F-actin or RhoA binding. We also demonstrate that DAAM1 depletion inhibits Golgi reorientation in wound healing assays. Wound-edge cells exhibit multiple protrusions characteristic of unpolarized cell migration. Finally, in U2OS cells lines stably expressing DAAM1, we observe an enhanced myosin IIB stress fiber network which opposes cell migration. CONCLUSIONS/SIGNIFICANCE: This work highlights the importance of DAAM1 in processes underlying cell polarity and suggests that it acts in part by affecting the function of acto-myosin IIB system. It also emphasizes the importance of the N-terminal half of DAAM1. DAAM1 depletion strongly blocks centrosomal re-polarization, supporting the concept that DAAM1 signaling cooperates with the established Cdc42 associated polarity complex. These findings are also consistent with the observation that ablation of myosin IIB but not myosin IIA results in polarity defects downstream of Wnt signaling. The structure-function analysis of DAAM1 in cultured cells parallels more complex morphological events in the developing embryo.