Table of contents

Rho associated kinases: ROCK and Citron

The small GTPase Rho induces the formation of actin stress fibers and mediates the formation of diverse actin structures. What remains unclear is how Rho regulates its effectors to elicit such functions. GTP-bound Rho activates its effector mDia1 by disrupting mDia1's intramolecular interactions. mDia1 protein is the first identified mammalian homolog of Drosophila Diaphanous, which is essential for cytokinesis, and belongs to a family of formin-homology (FH) proteins. FH proteins share structural features, including the tandemly aligned FH1 and FH2 domains in their carboxy-terminal halves. The FH1 domain contains repetitive polyproline sequences, which interact with an actin-monomer-binding protein, profilin. FH proteins regulate cellular morphogenic events, possibly in collaboration with profilin. Indeed, mDia1, profilin and RhoA have been found to co-localize in dynamic plasma-membrane structures such as phorbol-ester-induced membrane ruffles and phagocytic cups. Because the inactivation of Rho by microinjection of C3 exoenzyme inhibits such co-localization, endogenous Rho may regulate the subcellular assembly of filamentous actin (F-actin) through mDia1 signaling, although the type of actin structure formed under the control of Rho-mDia1 signaling has largely remained unknown. In addition, a genetic study of a Costa Rica family, members of which often develop deafness without other symptoms, has identified hDIA1 as a gene responsible for non-syndromic deafness. The protein encoded by this gene is the human counterpart of mDia1. In the affected hDIA1 protein, 21 aberrant amino acids are substituted for the C-terminal 52 amino acids. It remains unknown how this C-terminal truncation induces pathogenicity and why it is inherited as a dominant trait (Watanabe, 1999 and references therein).

Active mDia1 induces the formation of thin actin stress fibers, which are disorganized in the absence of activity of the Rho-associated kinase ROCK. Moreover, active mDia1 transforms ROCK-induced condensed actin fibers into structures reminiscent of Rho-induced stress fibers. Thus mDia1 and ROCK work concurrently during Rho-induced stress-fiber formation. Intriguingly, mDia1 and ROCK, depending on the balance of the two activities, induce actin fibers of various thicknesses and densities. Thus Rho may induce the formation of different actin structures affected by the balance between mDia1 and ROCK signaling (Watanabe, 1999).

p160ROCK is a serine/threonine protein kinase that binds selectively to GTP-Rho; when bound, it becomes active. To identify its function, HeLa cells were transfected with wild type and mutants of p160ROCK and the morphology of the transfected cells were examined. Transfection with wild type and mutants containing the kinase domain and the coiled-coil forming region induces focal adhesions and stress fibers, while no induction is observed with a kinase-defective mutant or a mutant containing only the kinase domain. Rho-induced formation of focal adhesions and stress fibers is inhibited by co-expression of a mutant defective in both kinase and Rho-binding activities. Rho, however, still induces an increase in F-actin content in these cells. These results suggest that p160ROCK works downstream of Rho to induce formation of focal adhesions, and that Rho-induced actin polymerization is mediated by other effector(s) (Ishizaki, 1997).

The Ras-related GTPase RhoA controls signaling processes required for cytoskeletal reorganization, transcriptional regulation, and transformation. The ability of RhoA mutants to transform cells correlates not with transcription but with their ability to bind ROCK-I, an effector kinase involved in cytoskeletal reorganization. A recently developed specific ROCK inhibitor, Y-27632, and ROCK truncation mutants were used to investigate the role of ROCK kinases in transcriptional activation and transformation. In NIH3T3 cells, Y-27632 does not prevent the activation of serum response factor, transcription of c-fos or cell cycle re-entry following serum stimulation. Repeated treatment of NIH3T3 cells with Y-27632, however, substantially disrupts their actin fiber network but does not affect their growth rate. Y-27632 blocks focus formation by activated RhoA and its guanine-nucleotide exchange factors Dbl and mNET1. It does not affect the growth rate of cells transformed by Dbl and mNET1, but restores normal growth control at confluence and prevents their growth in soft agar. Y-27632 also significantly inhibits focus formation by Ras, but has no effect on the establishment or maintenance of transformation by Src. Furthermore, it significantly inhibits anchorage-independent growth of two out of four colorectal tumor cell lines. Consistent with these data, a truncated ROCK derivative exhibits weak ability to cooperate with activated Raf in focus formation assays. It is concluded that ROCK signaling is required for both the establishment and maintenance of transformation by constitutively activated RhoA, and that it contributes to the Ras-transformed phenotype. These observations provide a potential explanation for the requirement for Rho in Ras-mediated transformation. Moreover, the inhibition of ROCK kinases may be of therapeutic use (Sahai, 1999).

The small GTPase RhoA plays a critical role in signaling pathways activated by serum-derived factors, such as lysophosphatidic acid (LPA), including the formation of stress fibers in fibroblasts and neurite retraction and rounding of soma in neuronal cells. Ectopic expression of v-Crk (an SH2/SH3 domain, containing adapter proteins) potentiates nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells and promotes the survival of cells when NGF is withdrawn. When cultured in 15% serum or lysophosphatidic acid-containing medium, the majority of v-Crk-expressing PC12 cells (v-CrkPC12 cells) display a flattened phenotype with broad lamellipodia and are refractory to NGF-induced neurite outgrowth unless serum is withdrawn. v-Crk-mediated cell flattening is inhibited by treatment of cells with C3 toxin or by mutation in the Crk SH2 or SH3 domain. Transient cotransfection of 293T cells with expression plasmids for p160ROCK (Rho-associated coiled-coil-containing kinase) and v-Crk, but not SH2 or SH3 mutants of v-Crk, results in hyperactivation of p160ROCK. Moreover, the level of phosphatidylinositol-4,5-bisphosphate is increased in v-CrkPC12 cells, as compared to the levels in mutant v-Crk-expressing cells or wild-type cells, consistent with PI(4)P5 kinase being a downstream target for Rho. Expression of v-Crk in PC12 cells does not result in activation of Rac- or Cdc42-dependent kinases PAK and S6 kinase, demonstrating specificity for Rho. In contrast to native PC12 cells, in which focal adhesions and actin stress fibers are not observed, immunohistochemical analysis of v-CrkPC12 cells reveals focal adhesion complexes that are formed at the periphery of the cell and are connected to actin cables. The formation of focal adhesions correlates with a concomitant upregulation in the expression of focal adhesion proteins FAK, paxillin, alpha3-integrin, and a higher-molecular-weight form of beta1-integrin. These results indicate that v-Crk activates the Rho-signaling pathway and serves as a scaffolding protein during the assembly of focal adhesions in PC12 cells (Altun-Gultekin, 1998).

The actin cytoskeleton undergoes extensive remodeling during cell morphogenesis and motility. The small guanosine triphosphatase Rho regulates such remodeling, but the underlying mechanisms of this regulation remain unclear. Cofilin exhibits actin-depolymerizing activity that is inhibited as a result of its phosphorylation by LIM-kinase. Cofilin is phosphorylated in N1E-115 neuroblastoma cells during lysophosphatidic acid-induced, Rho-mediated neurite retraction. This phosphorylation is sensitive to Y-27632, a specific inhibitor of the Rho-associated kinase ROCK. ROCK, which is a downstream effector of Rho, does not phosphorylate cofilin directly but phosphorylates LIM-kinase, which in turn is activated in order to phosphorylate cofilin. Overexpression of LIM-kinase in HeLa cells induces the formation of actin stress fibers in a Y-27632-sensitive manner. These results indicate that phosphorylation of LIM-kinase by ROCK and consequently increased phosphorylation of cofilin by LIM-kinase contribute to Rho-induced reorganization of the actin cytoskeleton (Maekawa, 1999).

Previous studies have shown that LIM-kinase activation and cofilin phosphorylation occur in response to Rac, another member of the Rho family GTPases, but not to Rho. It was determined that activation of Rho results in cofilin phosphorylation by LIM-kinase in vivo by expressing constitutively active V14-Rho and tagged cofilin in COS-7 cells. This experiment was done under conditions in which the activity of endogenous Rac was inhibited by expression of the dominant negative mutant N17-Rac, and that of endogenous Rho was inhibited by culture in serum-free medium. Expression of N17-Rac almost completely abolishes the cofilin phosphorylation observed in cells expressing tagged cofilin alone, suggesting that endogenous Rac is activated under basal conditions and induces cofilin phosphorylation. In the presence of N17-Rac, V14-Rho increases cofilin phosphorylation in transfected cells, and this increase is inhibited by coexpression of a dominant negative ROCK mutant, ROCK-KDIA. These observations suggest that the Rho-ROCK pathway is linked to cofilin phosphorylation in vivo, and that the previous studies may have masked this pathway by activation of endogenous Rac and Rho. The link between Rho-ROCK signaling and the LIM-kinase-cofilin pathway was investigated at the morphological level. Overexpression of LIMK1 in HeLa cells induces the formation of thick, bundled stress fibers that resemble those induced by active Rho or ROCK. Incubation of the transfected cells with Y-27632 results in the dissolution of these fibers, indicating that LIM-kinase collaborates with the Rho-ROCK pathway to induce stress fiber formation (Maekawa, 1999 and references).

The small GTP-binding protein Rho functions as a molecular switch in the formation of focal adhesions, stress fibers, cytokinesis and transcriptional activation. The biochemical mechanism underlying these actions remains unknown. Using a ligand overlay assay, a 160 kDa platelet protein has been isolated that binds specifically to GTP-bound Rho. This protein, p160, undergoes autophosphorylation at its serine and threonine residues and shows the kinase activity to exogenous substrates. Both activities are enhanced by the addition of GTP-bound Rho. A cDNA encoding p160 codes for a 1354 amino acid protein. This protein has a Ser/Thr kinase domain at its N-terminus, followed by a coiled-coil structure approximately 600 amino acids long, and at the C-terminus, a cysteine-rich zinc finger-like motif and a pleckstrin homology region. The N-terminus region including a kinase domain; a part of the coiled-coil structure shows strong homology to myotonic dystrophy kinase over 500 residues. When co-expressed with RhoA in COS cells, p160 is co-precipitated with the expressed Rho and its kinase activity is activated, indicating that p160 can associate physically and functionally with Rho both in vitro and in vivo (Ishizaki, 1996).

A novel serine/threonine kinase belonging to the myotonic dystrophy kinase family has been identified. The kinase can be produced in at least two different isoforms: an approximately 240-kDa protein (Citron Rho-interacting kinase, CRIK: see Drosophila Citron), in which the kinase domain is followed by the sequence of Citron, and a previously identified Rho/Rac binding protein, and an approximately 54-kDa protein [CRIK-short kinase (SK)], which consists mostly of the kinase domain. CRIK and CRIK-SK proteins are capable of phosphorylating exogenous substrates as well as of autophosphorylation, when tested by in vitro kinase assays after expression into COS7 cells. CRIK kinase activity is increased severalfold by coexpression of constitutively active Rho, while active Rac has more limited effects. Kinase activity of endogenous CRIK is indicated by in vitro kinase assays after immunoprecipitation with antibodies recognizing the Citron moiety of the protein. When expressed in keratinocytes, full-length CRIK, but not CRIK-SK, localizes into corpuscular cytoplasmic structures and elicits recruitment of actin into these structures. The previously reported Rho-associated kinases ROCK I and II are ubiquitously expressed. In contrast, CRIK exhibits a restricted pattern of expression, suggesting that this kinase may fulfill a more specialized function in specific cell types (Di Cunto, 1998).

Synaptic NMDA-type glutamate receptors are anchored to the second of three PDZ (PSD-95/Discs large/ZO-1) domains in the postsynaptic density (PSD) protein PSD-95. Citron, a protein target for the activated form of the small GTP-binding protein Rho, preferentially binds the third PDZ domain of PSD-95. In GABAergic neurons from the hippocampus, citron forms a complex with PSD-95 and is concentrated at the postsynaptic side of glutamatergic synapses. Citron is expressed only at low levels in glutamatergic neurons in the hippocampus and is not detectable at synapses onto these neurons. In contrast to citron, both p135 SynGAP (an abundant synaptic Ras GTPase-activating protein that can bind to all three PDZ domains of PSD-95) and Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) are concentrated postsynaptically at glutamatergic synapses on glutamatergic neurons. SynGAP, a Ras GTPase activating protein, is nearly as abundant in the PSD fraction as PSD-95 itself. SynGAP can be phosphorylated by Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) in the PSD fraction and its GAP activity is reduced after phosphorylation. Thus, SynGAP and CaM kinase II constitute a signal transduction complex associated with the NMDA receptor. CaM kinase II is not expressed and p135 SynGAP is expressed in less than half of hippocampal GABAergic neurons. Segregation of citron into inhibitory neurons does not occur in other brain regions. For example, citron is expressed at high levels in most thalamic neurons, which are primarily glutamatergic and contain CaM kinase II. In several other brain regions, citron is present in a subset of neurons that can be either GABAergic or glutamatergic and can sometimes express CaM kinase II. Thus, in the hippocampus, signal transduction complexes associated with postsynaptic NMDA receptors are different in glutamatergic and GABAergic neurons and are specialized in a way that is specific to the hippocampus (Zhang, 1999).

The results presented here support the notion that differential expression of PSD-95-binding proteins in different neurons helps to determine the composition of signal transduction complexes formed by association with PSD-95 at glutamatergic PSDs. The resulting distinct compositions of these complexes will likely define the nature of local biochemical signaling associated with activation of NMDA receptors. The selective localization of citron suggests that, in hippocampus, PSDs of glutamatergic synapses made onto inhibitory interneurons contain cytoskeletal regulatory machinery that is not present at glutamatergic synapses made onto excitatory principal neurons. Furthermore, CaM kinase II is not detectable in these same PSDs but is present in the postsynaptic complex of excitatory synapses made onto glutamatergic neurons in the hippocampus. CaM kinase II can phosphorylate and regulate the GluRA/1 subunit of AMPA-type glutamate receptors and the synaptic Ras GTPase-activating protein SynGAP and can phosphorylate the NR2A and NR2B subunits of the NMDA receptor. This regulation by CaM kinase II is absent from the postsynaptic side of glutamatergic synapses on hippocampal inhibitory neurons. Thus, the modes of regulation of synaptic structure (by citron) and of synaptic strength (by CaM kinase II or citron) at glutamatergic synapses will differ dramatically between excitatory and inhibitory neurons. High citron expression found only in GABAergic neurons appears to be a unique feature of the hippocampus. In other brain regions, such as the thalamus and cerebral cortex, citron and CaM kinase II are often found together in excitatory neurons. Thus, the composition of signal transduction machinery at the postsynaptic membrane of glutamatergic synapses varies among neurons throughout the brain in ways that cannot be classified simply. Furthermore, findings regarding the mechanisms of signal transduction and plasticity at hippocampal synapses may not always generalize to synapses in other areas of the brain (Zhang, 1999).

Proteins of the membrane-associated guanylate kinase family play an important role in the anchoring and clustering of neurotransmitter receptors in the postsynaptic density (PSD) at many central synapses. However, relatively little is known about how these multifunctional scaffold proteins might provide a privileged site for activity- and cell type-dependent specification of the postsynaptic signaling machinery. Classically, the Rho signaling pathway has been implicated in mechanisms of axonal outgrowth, dendrogenesis, and cell migration during neural development, but its contribution remains unclear at the synapses in the mature CNS. Evidence is presented that Citron, a Rho-effector in the brain, is enriched in the PSD fraction and interacts with PSD-95/synapse-associated protein (SAP)-90 both in vivo and in vitro. Citron colocalization with PSD-95 occurs, not exclusively but certainly, at glutamatergic synapses in a limited set of neurons, such as the thalamic excitatory neurons; Citron expression, however, cannot be detected in the principal neurons of the hippocampus and the cerebellum in the adult mouse brain. In a heterologous system, Citron was shown to form a heteromeric complex not only with PSD-95 but also with NMDA receptors. Thus, Citron-PSD-95/SAP-90 interaction may provide a region- and cell type-specific link between the Rho signaling cascade and the synaptic NMDA receptor complex (Furuyashiki, 1999).

The contributions of the small GTPase Rho and its downstream target p160ROCK during the early stages of axon formation were tested in cultured cerebellar granule neurons. p160ROCK inhibition (by use of a drug targeting ROCK or by use of dominant negative ROCK), triggers immediate outgrowth of membrane ruffles and filopodia (presumably by reducing the stability of the cortical actin network), followed by the generation of initial growth cone-like membrane domains from which axonal processes arise. Furthermore, a potentiation in both the size and the motility of growth cones is evident, though the overall axon elongation rate remains stable. Conversely, it is suggested that overexpression of dominant active forms of Rho or ROCK prevents initiation of axon outgrowth. Taken together, these data indicate a novel role for the Rho/ROCK pathway as a gate critical for the initiation of axon outgrowth and the control of growth cone dynamics (Bito, 2000).

Transfection experiments using wild-type and a kinase-dead mutant LIM kinase-1 (LIMK-1) suggest an involvement of LIMK-1 as a downstream target of ROCK in the regulation of axon formation. It is speculated that during the initial stages of axon outgrowth, the activation of the Rho/ROCK/LIMK-dependent phosphorylation cascade strongly contributes to maintaining an inactivation state of cofilin and preventing it from being activated by dephosphorylation. When this phosphorylation is counteracted, such as by use of ROCK inhibitor Y-27632 in vitro, or by Rho inhibition in vivo, local accumulation of activated cofilin may indeed trigger a rapid morphological change and generation of neuronal polarity in a Rho-regulated manner (Bito, 2000).

The epidermis comprises multiple layers of specialized epithelial cells called keratinocytes. As cells are lost from the outermost epidermal layers, they are replaced through terminal differentiation, in which keratinocytes of the basal layer cease proliferating, migrate upwards, and eventually reach the outermost cornified layers. Normal homeostasis of the epidermis requires that the balance between proliferation and differentiation be tightly regulated. The GTP binding protein RhoA plays a fundamental role in the regulation of the actin cytoskeleton and in the adhesion events that are critically important to normal tissue homeostasis. Two central mediators of the signals from RhoA are the ROCK serine/threonine kinases ROCK-I and ROCK-II. ROCK's role in the regulation of epidermal keratinocyte function was examined by using a pharmacological inhibitor and expressing conditionally active or inactive forms of ROCK-II in primary human keratinocytes. vlocking ROCK function results in inhibition of keratinocyte terminal differentiation and an increase in cell proliferation. In contrast, activation of ROCK-II in keratinocytes results in cell cycle arrest and an increase in the expression of a number of genes associated with terminal differentiation. Thus, these results indicate that ROCK plays a critical role in regulating the balance between proliferation and differentiation in human keratinocytes (McMullan, 2004).

CDK inhibitor p27 interacts with RhoA to modulate cell migration

The tumor suppressor p27Kip1 is an inhibitor of cyclin/cyclin-dependent kinase (CDK) complexes and plays a crucial role in cell cycle regulation. However, p27Kip1 also has cell cycle-independent functions. Indeed, p27Kip1 regulates cell migration, as evidenced by the observation that p27Kip1-null fibroblasts exhibit a dramatic decrease in motility compared with wild-type cells. The regulation of motility by p27Kip1 is independent of its cell-cycle regulatory functions; re-expression of both wild-type p27Kip1 and a mutant p27Kip1 (p27CK) that cannot bind to cyclins and CDKs rescues migration of p27–/– cells. p27–/– cells have increased numbers of actin stress fibers and focal adhesions. This is reminiscent of cells in which the Rho pathway is activated. Indeed, active RhoA levels were increased in cells lacking p27Kip1. Moreover, inhibition of ROCK, a downstream effector of Rho, is able to rescue the migration defect of p27–/– cells in response to growth factors. Finally, p27Kip1 is found to bind to RhoA, thereby inhibiting RhoA activation by interfering with the interaction between RhoA and its activators, the guanine–nucleotide exchange factors (GEFs). Together, the data suggest a novel role for p27Kip1 in regulating cell migration via modulation of the Rho pathway (Besson, 2004).

It appears that all of the members of the Cip/Kip family of CKIs may play a role in the regulation of the Rho pathway, albeit acting at distinct levels in the pathway. p27Kip1 has been shown in this study to regulate Rho activation. Cytoplasmic p21Waf1/Cip1 has been shown to directly inhibit ROCK, resulting in increased neurite outgrowth (Tanaka, 2002; Lee, 2003). However, in the current experiments the loss of p21Waf1 in MEFs had no effect on their migratory ability. p57Kip2 binds to LIM-kinase and induces its translocation to the nucleus, thereby inhibiting its activity (Yokoo, 2003). LIM-kinase phosphorylates and inactivates the actin depolymerization factor cofilin, and is itself directly activated by ROCK phosphorylation. Therefore, although they act differently, all of the Cip/Kip proteins seem to negatively regulate the Rho signaling pathway when in the cytoplasm. It is tempting to speculate that the regulation of different proteins in the Rho pathway by CKIs could provide new levels of regulation of Rho-mediated processes, because the abundance and subcellular localization of CKIs are regulated throughout the cell cycle (Besson, 2004).

Table of contents

Rho1: Biological Overview | Regulation | Protein Interactions | Developmental Biology | Effects of Mutation | References

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