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Protein tyrosine kinase Pyk2 and multifunctional adaptor protein Cbl are implicated in the regulation of the cytoskeleton in several cell types. We report that Pyk2 and Cbl form a signaling complex that is translocated to lipid rafts and is enriched in growth cones of differentiating PC12 cells following growth factor stimulation. We found that Pyk2 and Cbl interacted with the adaptor protein ArgBP2, which also bound to flotillin-1, a component of lipid raft microdomains. These interactions contributed to recruitment of the Pyk2/Cbl complex to lipid raft compartments. In addition, Pyk2, Cbl and ArgBP2 were found co-localized with actin in axons and growth cones of differentiated PC12 cells. Moreover, co-expression of Pyk2, ArgBP2 and Cbl facilitated growth factor-induced formation of lamellipodia at the tip of neurites. Formation of these growth cone lamellipodia was dependent on intact lipid rafts and the Cbl-associated effectors Crk and phosphatidylinositol 3 (PI 3)-kinase. Our results indicate that recruitment of Pyk2/Cbl complexes to lipid rafts participates in growth factor-induced regulation of the actin cytoskeleton in growing neurites. [ABSTRACT FROM AUTHOR]

Integrins play a pivotal role in serf-regulated hematopoietic adhesion and migration. Leukocyte αvβ3 integrimmediated adhesion to vitronectin requires protein kinase C activation and phosphorylation on tyrosine 747 of the β3 cytoplasmic tail. We have previously shown that β3 phosphorylation is required for Rho activation. In this study, an antibody specific to phosphorylated β3 tyrosine 747 was used to localize phosphorylated αvβ3 in vitronectin adhesive structures. Early adhesion contacts containing phosphorylated β3 preceded actin stress fiber formation. β3 phosphorylation decreased progressively throughout the course of adhesion coincident with the appearance of actin stress fibers. Time-dependent increases in colocalization of β3 with tyrosine 402 phosphorylated Pyk2 in similar adhesive structures was observed, providing evidence for downstream signaling complex formation. Surprisingly, Arp3 organized into similar adhesion contacts in cells expressing wild-type β3 but not in those expressing a nonphosphorylatable mutant of β3, suggesting that β3 phosphorylation is required for sequestration of Arp3 to adhesion complexes. Suppression of actin stress fiber formation by an inhibitor to Rho kinase disrupted Arp3 organization while prolonging β3 phosphorylation throughout the adhesion time course. These data confirm a requirement for β3 phosphorylation in αvβ3-mediated adhesion to vitronectin and suggest that β3 phosphorylation permits signaling complex assembly at the adhesion site necessary for actin stress fiber formation in leukocytes. [ABSTRACT FROM AUTHOR]

STIM1- and Orai1-mediated store-operated Ca 2+ entry (SOCE) constitutes the major Ca 2+ influx in almost all electrically non-excitable cells. However, little is known about the spatiotemporal organization at the elementary level. Here, we developed Orai1-tethered or palmitoylated biosensor GCaMP6f to report subplasmalemmal Ca 2+ signals. We visualized spontaneous discrete and long-lasting transients ('Ca 2+ glows') arising from STIM1-Orai1 in invading melanoma cells. Ca 2+ glows occurred preferentially in single invadopodia and at sites near the cell periphery under resting conditions. Re-addition of external Ca 2+ after store depletion elicited spatially synchronous Ca 2+ glows, followed by high-rate discharge of asynchronous local events. Knockout of STIM1 or expression of the dominant-negative Orai1-E106A mutant markedly decreased Ca 2+ glow frequency, diminished global SOCE and attenuated invadopodial formation. Functionally, invadopodial Ca 2+ glows provided high Ca 2+ microdomains to locally activate Ca 2+ /calmodulin-dependent Pyk2 (also known as PTK2B), which initiates the SOCE-Pyk2-Src signaling cascade required for invasion. Overall, the discovery of elemental Ca 2+ signals of SOCE not only unveils a previously unappreciated gating mode of STIM1-Orai1 channels in situ , but also underscores a critical role of the spatiotemporal dynamics of SOCE in orchestrating complex cell behaviors such as invasion. This article has an associated First Person interview with the first author of the paper., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (Pyk2) are related tyrosine kinases that have important cellular functions, primarily through regulation of the cytoskeleton. Recent studies have identified multiple molecular mechanisms that regulate cytoskeletal responses, and have provided important and exciting insights into how FAK and Pyk2 control cellular processes such as cell migration. Equally exciting are reports of novel and originally unanticipated functions of these kinases, providing the groundwork for future avenues of investigation. This Commentary summarizes some of these recent discoveries that are relevant to the control of biological responses of the cell. [ABSTRACT FROM AUTHOR]