Dynamic Regulation of Src by Csk in Integrin α IIb β 3 Mediated Signalling.

When platelets adhere to fibrinogen, they undergo profound actin rearragements and spreading. These responses require signals triggered by aIIbb3, a process known as outside-in signalling. Although aIIbb3 signaling is required for normal hemostasis, its molecular basis is incompletely understood. Recently we demonstrated by coimmunoprecipitation techniques that several Src family kinases are constitutively associated with aIIbb3 in platelets. Fibrinogen binding to aIIbb3 leads to rapid activation of Src and recruitement and activation of the Syk tyrosine kinase. Theses interactions are significant because murine platelets defiecient in multiple Src kinases or Syk fail to spread on fibrinogen. One of the earliest events upon binding of fibrinogen to the receptor is the assembly of a multimolecular protein complex inculding the tyrosine kinases Src and Syk. Phosphorylation of Src at Tyrosine 418 leads to Src activation, phosphorylation of Tyrosine 529 to inactivation. The inactivation of Src is mediated by Csk. Previous studies have shown that in resting platelets Csk forms a complex with integrin α IIb β3 and Src. After fibrinogen binding Csk leaves this complex.

The aim of this study is to elucidate the dynamics of Src inactivation and the interaction with Csk in this process by using Fluorescence Resonance Energy Transfer (FRET). A5 CHO cells stably expressing integrin α IIb β3 were transfected with Csk-YFP and various Src mutatnts in a GFP vector. After cell adhesion on fibrinogen, a strong FRET signal could be observed at the edges of nascent lamellipodia of the spreading cell. The FRET signal was dynamic since it was transported in waves inside the cell within seconds. After completion of the adhesion process a weak FRET signal remained visible inside the cell. A different pattern could be observed when cells were transfected with Csk and the active Src mutant Y529F. The observed FRET signal was much weaker and less dynamic after cells were allowed to spread on fibrinogen suggesting that the muation of Tyrosine 529 to Phenylalanine inhibited Csk binding and inactivation of Src. This was not due to a spreading defect since the cells appeared to spread on fibrinogen normally. The use of the inactive Src mutant K295R revealed a FRET signal comparable to WT Src. These studies suggest that Src inactivation is dynamically regulated within seconds after cell adhesion on fibrinogen and that Tyrosine 529 is required for the interaction of Csk with Src in this process.