Abstract
In vascular smooth muscle (VSM) cells, Ca(2+)/calmodulin-dependent protein kinase IIδ2 (CaMKIIδ2) activates non-receptor tyrosine kinases and EGF receptor, with a Src family kinase as a required intermediate. siRNA-mediated suppression of Fyn, a Src family kinase, inhibited VSM cell motility. Simultaneous suppression of both Fyn and CaMKIIδ2 was non-additive, suggesting coordinated regulation of cell motility. Confocal immunofluorescence microscopy indicated that CaMKIIδ2 and Fyn selectively (compared with Src) co-localized with the Golgi in quiescent cultured VSM cells. Stimulation with PDGF resulted in a rapid (<5 min) partial redistribution and co-localization of both kinases in peripheral membrane regions. Furthermore, CaMKIIδ2 and Fyn selectively (compared with Src) co-immunoprecipitated, suggesting a physical interaction in a signaling complex. Stimulation of VSM cells with ionomycin, a calcium ionophore, resulted in activation of CaMKIIδ2 and Fyn and disruption of the complex. Pretreatment with KN-93, a pharmacological inhibitor of CaMKII, prevented activation-dependent disruption of CaMKIIδ2 and Fyn, implicating CaMKIIδ2 as an upstream mediator of Fyn. Overexpression of constitutively active CaMKII resulted in the dephosphorylation of Fyn at Tyr-527, which is required for Fyn activation. Taken together, these data demonstrate a dynamic interaction between CaMKIIδ2 and Fyn in VSM cells and indicate a mechanism by which CaMKIIδ2 and Fyn may coordinately regulate VSM cell motility.
Highlights
Increased vascular smooth muscle cell motility results in neointimal formation
SFKs Mediate vascular smooth muscle (VSM) Cell Migration—SFKs are multifunctional non-receptor tyrosine kinases that have been implicated in various cell functions, including migration [14]
To confirm a general function of SFKs in VSM cell migration, VSM cells were treated with 3 M PP2, a pharmacological inhibitor of SFKs [26], prior to scratch wounding a monolayer of VSM cells as described under “Experimental Procedures.”
Summary
Increased vascular smooth muscle cell motility results in neointimal formation. Results: CaMKII␦2 and Fyn physically interact, and CaMKII␦2 activity regulates complex formation, Fyn activity, and motility. Overexpression of constitutively active CaMKII resulted in the dephosphorylation of Fyn at Tyr527, which is required for Fyn activation Taken together, these data demonstrate a dynamic interaction between CaMKII␦2 and Fyn in VSM cells and indicate a mechanism by which CaMKII␦2 and Fyn may coordinately regulate VSM cell motility. We show by co-localization and immunoprecipitation that CaMKII␦2 interacts selectively with Fyn compared with other SFKs and that CaMKII␦2 regulates tyrosine phosphorylation events required for Fyn activity. These results provide a potential mechanism by which CaMKII␦2 and Fyn coordinately regulate VSM cell motility
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