Clinton Monfries

α2-Chimaerin, Cyclin-Dependent Kinase 5/p35, and Its Target Collapsin Response Mediator Protein-2 Are Essential Components in Semaphorin 3A-Induced Growth-Cone Collapse

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)α2-chimaerin is involved in Semaphorin 3A (Sema 3A) signaling. In dorsal root ganglion neurons, Sema 3A-induced growth cone collapse was inhibited by α2-chimaerin mutated to eliminate GAP activity or interaction with phosphotyrosine. Activation of α2-chimaerin by phorbol ester caused growth cone collapse. Active α2-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 3β (GSK3β), 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. α2-chimaerin, like CRMP-2, can associate with the Sema 3A receptor. These results indicate that active α2-chimaerin Rac GAP, Cdk5/p35, and its substrate CRMP-2, are implicated in the dynamics of growth cone guidance initiated through Sema 3A signaling.

Collapsin Response Mediator Protein Switches RhoA and Rac1 Morphology in N1E-115 Neuroblastoma Cells and Is Regulated by Rho Kinase

The formation and directional guidance of neurites involves dynamic regulation of Rho family GTPases. Rac and Cdc42 promote neurite outgrowth, whereas Rho activation causes neurite retraction. Here we describe a role for collapsin response mediator protein (Crmp-2), a neuronal protein implicated in axonal outgrowth and a component of the semaphorin 3A pathway, in switching GTPase signaling when expressed in combination with either dominant active Rac or Rho. In neuroblastoma N1E-115 cells, co-expression of Crmp-2 with dominant active RhoA V14 induced Rac morphology, cell spreading and ruffling (and the formation of neurites). Conversely, co-expression of Crmp-2 with dominant active Rac1 V12 inhibited Rac morphology, and in cells already expressing Rac1 V12, Crmp-2 caused localized peripheral collapse, involving Rho (and Cdc42) activation. Rho kinase was a pivotal regulator of Crmp-2; Crmp-2 phosphorylation was required for Crmp-2/Rac1 V12 inhibition, but not Crmp-2/RhoA V14 induction, of Rac morphology. Thus Crmp-2, regulated by Rho kinase, promotes outgrowth and collapse in response to active Rho and Rac, respectively, reversing their usual morphological effects and providing a mechanism for dynamic modulation of growth cone guidance.

Relationship of p21-activated kinase (PAK) and filopodia to persistence and oncogenic transformation

Previously, we found that oncogenically transformed cells had fewer filopodia and more large, p21‐activated kinase (PAK)‐dependent features than normal cells. These large protrusions (LPs) were increased in cells expressing RhoAN19 with Cdc42‐associated kinase (ACK). Here, we determine how GTPase‐mediated mechanisms of focal contact (FC) regulation affect these protrusions. Constructs encoding various proteins were introduced into cells which were then studied by microscopy and computerized image processing and analysis. Constructs that prevented PAK recruitment by PAK‐interacting exchange factor (PIX) or restricted PAK residence time on FCs decreased both protrusions. Thus, filopodia were also PAK‐dependent. A comparison of FC distribution in cells expressing PAK in the presence or absence of PAK kinase inhibitor domain (KID) suggested that PAK enlarged FCs without affecting the prevalence of either protrusion. KID or Nck expression increased LPs but not filopodia. Nck failed to synergize with KID or ACK and RhoAN19 in enhancing LPs. Nck and KID synergistically enhanced filopodia, possibly because Nck recruited PAK to FCs while KID prevented their dissociation by PAK‐mediated autophosphorylation. Coexpression of Nck, ACK, and RhoAN19 abrogated filopodia and replicated the transformed phenotype. Since Nck recruitment of PAK is implicated in persistence of directional movement, we studied the PAK–Nck interface. Filopodia were eliminated by the Nck PAK‐binding domain and LPs by the PAK Nck‐binding domain. The results suggested that filopodia formation has more stringent requirements than LP formation, and Nck and PAK are used differently in the protrusions. Loss of filopodia in transformed cells may reflect defective regulation of GTPase mechanisms. J. Cell. Physiol. 220: 576–585, 2009.