Katsuhiro Kato

PAR-6-PAR-3 mediates Cdc42-induced Rac activation through the Rac GEFs STEF/Tiam1

A polarity complex of PAR-3, PAR-6 and atypical protein kinase C (aPKC) functions in various cell-polarization events, including neuron specification. The small GTPase Cdc42 binds to PAR-6 and regulates cell polarity. However, little is known about the downstream signals of the Cdc42–PAR protein complex. Here, we found that PAR-3 directly interacted with STEF/Tiam1, which are Rac-specific guanine nucleotide-exchange factors, and that STEF formed a complex with PAR-3–aPKC–PAR-6–Cdc42-GTP. Cdc42 induces lamellipodia in a Rac-dependent manner in N1E-115 neuroblastoma cells. Disruption of Cdc42–PAR-6 or PAR-3–STEF binding inhibited Cdc42-induced lamellipodia but not filopodia. The isolated STEF-binding PAR-3 fragment was sufficient to induce lamellipodia independently of Cdc42 and PAR-6. PAR-3 is required for Cdc42-induced Rac activation, but is not essential for lamellipodia formation itself. In cultured hippocampal neurons, STEF accumulated at the tip of the growing axon and colocalized with PAR-3. The spatio-temporal activation and signalling of Cdc42–PAR-6–PAR-3–STEF/Tiam1–Rac seem to be involved in neurite growth and axon specification. We propose that the PAR-6–PAR-3 complex mediates Cdc42-induced Rac activation by means of STEF/Tiam1, and that this process seems to be required for the establishment of neuronal polarity.

Role of the PAR-3–KIF3 complex in the establishment of neuronal polarity

Neurons polarize to form elaborate multiple dendrites and one long axon. The establishment and maintenance of axon/dendrite polarity are fundamentally important for neurons. Recent studies have demonstrated that the polarity complex PAR-3–PAR-6–atypical protein kinase C (aPKC) is involved in polarity determination in many tissues and cells. The function of the PAR-3–PAR-6–aPKC protein complex depends on its subcellular localization in polarized cells. PAR-3 accumulates at the tip of growing axons in cultured rat hippocampal neurons, but the molecular mechanism of this localization remains unknown. Here we identify a direct interaction between PAR-3 and KIF3A, a plus-end-directed microtubule motor protein, and show that aPKC can associate with KIF3A through its interaction with PAR-3. The expression of dominant-negative PAR-3 and KIF3A fragments that disrupt PAR-3–KIF3A binding inhibited the accumulation of PAR-3 and aPKC at the tip of the neurites and abolished neuronal polarity. These results suggest that PAR-3 is transported to the distal tip of the axon by KIF3A and that the proper localization of PAR-3 is required to establish neuronal polarity.

CRMP-2 regulates polarized Numb-mediated endocytosis for axon growth

Axon growth during neural development is highly dependent on both cytoskeletal re-organization and polarized membrane trafficking. Previously, we demonstrated that collapsin response mediator protein-2 (CRMP-2) is critical for specifying axon/dendrite fate and axon growth in cultured hippocampal neurons, possibly by interacting with tubulin heterodimers and promoting microtubule assembly. Here, we identify Numb as a CRMP-2-interacting protein. Numb has been shown to interact with α-adaptin and to be involved in endocytosis. We found that Numb was associated with L1, a neuronal cell adhesion molecule that is endocytosed and recycled at the growth cone, where CRMP-2 and Numb were colocalized. Furthermore, expression of dominant-negative CRMP-2 mutants or knockdown of CRMP-2 message with small-interfering (si) RNA inhibited endocytosis of L1 at axonal growth cones and suppressed axon growth. These results suggest that in addition to regulating microtubule assembly, CRMP-2 is involved in polarized Numb-mediated endocytosis of proteins such as L1.

Rho-kinase Contributes to Sustained RhoA Activation through Phosphorylation of p190A RhoGAP

RhoA is transiently activated by specific extracellular signals such as endothelin-1 (ET-1) in vascular smooth muscle cells. RhoGAP negatively regulates RhoA activity: thus, RhoA becomes the GDP-bound inactive form afterward. Sustained activation of RhoA is induced with high doses of the extracellular signals and is implicated in certain diseases such as vasospasms. However, it remains largely unknown how prolonged activation of RhoA is induced. Here we show that Rho-kinase, an effector of RhoA, phosphorylated p190A RhoGAP at Ser1150 and attenuated p190A RhoGAP activity in COS7 cells. Binding of Rnd to p190A RhoGAP is thought to enhance its activation. Phosphorylation of p190A RhoGAP by Rho-kinase impaired Rnd binding. Stimulation of vascular smooth muscle cells with a high dose of ET-1 provoked sustained RhoA activation and p190A RhoGAP phosphorylation, both of which were prohibited by a Rho-kinase inhibitor. The phosphomimic mutation of p190A RhoGAP weakened Rnd binding and RhoGAP activities. Taken together, these results suggest that ET-1 induces Rho-kinase activation and subsequent phosphorylation of p190A RhoGAP, leading to prolonged RhoA activation.

Integrin β1 controls VE-cadherin localization and blood vessel stability.

Angiogenic blood vessel growth requires several distinct but integrated cellular activities. Endothelial cell sprouting and proliferation lead to the expansion of the vasculature and give rise to a highly branched, immature plexus, which is subsequently reorganized into a mature and stable network. Although it is known that integrin-mediated cell-matrix interactions are indispensable for embryonic angiogenesis, little is known about the function of integrins in different steps of vascular morphogenesis. Here, by investigating the integrin β1-subunit with inducible and endothelial-specific gene targeting in the postnatal mouse retina, we show that β1 integrin promotes endothelial sprouting but is a negative regulator of proliferation. In maturing vessels, integrin β1 is indispensable for proper localization of VE-cadherin and thereby cell-cell junction integrity. The sum of our findings establishes that integrin β1 has critical functions in the growing and maturing vasculature, and is required for the formation of stable, non-leaky blood vessels.

The Actin-Binding Protein Girdin and Its Akt-Mediated Phosphorylation Regulate Neointima Formation After Vascular Injury

Rationale: It is well established that the migration and proliferation of vascular smooth muscle cells (VSMCs) have major roles in the vascular remodeling process. Our previous study showed that the Akt substrate Girdin, which is expressed in VSMCs and endothelial cells, is essential for postnatal angiogenesis. However, the function of Girdin and its Akt-mediated phosphorylation in VSMCs and their in vivo roles in vascular remodeling remain to be elucidated. Objective: We investigated the function of Girdin and its Akt-mediated phosphorylation using cultured VSMCs and animal models of vascular remodeling. Methods and Results: The depletion of Girdin by RNA interference disrupted the rearrangement of the actin cytoskeleton in VSMCs, resulting in impaired cell migration. The depletion of Girdin also inhibited VSMC proliferation. Girdin expression was highly upregulated and its serine at position 1416 was phosphorylated in the neointima of carotid arteries after balloon injury in a rat model. The introduction of an adenovirus harboring short hairpin RNA against Girdin attenuated the proliferation of VSMCs and neointima formation without affecting reendothelialization. Furthermore, we found that neointima formation after femoral wire injury was significantly attenuated in Girdin S1416A knock-in mice, in which the Akt phosphorylation site of Girdin was mutated, thus indicating a major role for Girdin phosphorylation in vascular remodeling. Conclusions: These findings indicate that Girdin and its Akt-mediated phosphorylation have major roles in the migration and proliferation of VSMCs and vascular remodeling, making the Akt/Girdin signaling pathway a potential target for the development of new therapeutics for vascular diseases.

Involvement of Girdin in the Determination of Cell Polarity during Cell Migration

Cell migration is a critical cellular process that determines embryonic development and the progression of human diseases. Therefore, cell- or context-specific mechanisms by which multiple promigratory proteins differentially regulate cell migration must be analyzed in detail. Girdin (girders of actin filaments) (also termed GIV, Gα-interacting vesicle associated protein) is an actin-binding protein that regulates migration of various cells such as endothelial cells, smooth muscle cells, neuroblasts, and cancer cells. Here we show that Girdin regulates the establishment of cell polarity, the deregulation of which may result in the disruption of directional cell migration. We found that Girdin interacts with Par-3, a scaffolding protein that is a component of the Par protein complex that has an established role in determining cell polarity. RNA interference-mediated depletion of Girdin leads to impaired polarization of fibroblasts and mammary epithelial cells in a way similar to that observed in Par-3-depleted cells. Accordingly, the expression of Par-3 mutants unable to interact with Girdin abrogates cell polarization in fibroblasts. Further biochemical analysis suggests that Girdin is present in the Par protein complex that includes Par-3, Par-6, and atypical protein kinase C. Considering previous reports showing the role of Girdin in the directional migration of neuroblasts, network formation of endothelial cells, and cancer invasion, these data may provide a specific mechanism by which Girdin regulates cell movement in biological contexts that require directional cell movement.

The inositol 5-phosphatase SHIP2 is an effector of RhoA and is involved in cell polarity and migration

Polarization in motile cells requires the coordination of several key signaling molecules, including RhoA small GTPases and phosphoinositides. It is found that SHIP2 interacts with RhoA in a GTP-dependent manner and this interaction is required for proper localization of PI(3,4,5)P3 and regulation of cell polarization and migration.

A proteomic approach for comprehensively screening substrates of protein kinases such as Rho-kinase.

Background Protein kinases are major components of signal transduction pathways in multiple cellular processes. Kinases directly interact with and phosphorylate downstream substrates, thus modulating their functions. Despite the importance of identifying substrates in order to more fully understand the signaling network of respective kinases, efficient methods to search for substrates remain poorly explored. Methodology/Principal Findings We combined mass spectrometry and affinity column chromatography of the catalytic domain of protein kinases to screen potential substrates. Using the active catalytic fragment of Rho-kinase/ROCK/ROK as the model bait, we obtained about 300 interacting proteins from the rat brain cytosol fraction, which included the proteins previously reported as Rho-kinase substrates. Several novel interacting proteins, including doublecortin, were phosphorylated by Rho-kinase both in vitro and in vivo. Conclusions/Significance This method would enable identification of novel specific substrates for kinases such as Rho-kinase with high sensitivity.

Kinase-interacting substrate screening is a novel method to identify kinase substrates

A novel method called kinase-interacting substrate screening based on affinity beads coated with the kinase of interest identifies phosphorylation sites for Rho-kinase and others, which reveals that Rho-kinase substrate Scrib plays a crucial role in the regulation of subcellular contractility by assembling with Rho-kinase and Shroom2.