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Activation of Rho-dependent cell spreading and focal adhesion biogenesis by the v-Crk adaptor protein
Z F Altun-Gultekin1, S Chandriani, C Bougeret
1Department of Neurology and Neuroscience, Cornell University Medical College, New York, New York, USA.
Abstract:
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. Previously, we have shown that ectopic expression of v-Crk, an SH2/SH3 domain-containing adapter proteins, in PC12 cells potentiates nerve growth factor (NGF)-induced neurite outgrowth and promotes the survival of cells when NGF is withdrawn. In the present study we show that, 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a flattened phenotype with broad lamellipodia and are refractory to NGF-induced neurite outgrowth unless serum is withdrawn.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 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 which 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. Our 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.
Insights
The adapter protein v-Crk activates the RhoA signaling pathway, leading to cell flattening and focal adhesion assembly in PC12 cells. This activation is specific to Rho and influences downstream targets like PI(4)P5 kinase.
Area of Science:
- Cell Biology
- Molecular Signaling
- Neuroscience
Background:
- RhoA GTPase is crucial for cellular processes like stress fiber formation and neurite retraction.
- v-Crk, an adapter protein, enhances nerve growth factor (NGF)-induced neurite outgrowth and cell survival.
- PC12 cells are a model system for neuronal differentiation and signaling studies.
Purpose of the Study:
- To investigate the role of v-Crk in PC12 cell morphology and signaling.
- To determine the specific Rho GTPase pathway activated by v-Crk.
- To elucidate the mechanism of v-Crk-mediated focal adhesion formation.
Main Methods:
- PC12 cells expressing v-Crk were cultured in serum or lysophosphatidic acid (LPA) medium.
- Cells were treated with C3 toxin or expressed v-Crk mutants.
- Co-transfection assays with p160ROCK and v-Crk were performed.
- Levels of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) and downstream kinases were analyzed.
- Immunohistochemistry was used to examine focal adhesions and associated proteins.
Main Results:
- v-Crk expression induced cell flattening and lamellipodia formation, refractory to NGF unless serum was withdrawn.
- v-Crk-mediated flattening was inhibited by C3 toxin and v-Crk SH2/SH3 domain mutations.
- v-Crk hyperactivated p160ROCK and increased PI(4,5)P2 levels, indicating Rho pathway activation.
- v-Crk specifically activated Rho, not Rac or Cdc42, and promoted focal adhesion assembly with associated protein upregulation.
Conclusions:
- v-Crk activates the Rho signaling pathway in PC12 cells.
- v-Crk acts as a scaffolding protein in the assembly of focal adhesions.
- Rho pathway activation by v-Crk influences cell morphology and cytoskeletal organization.