Dynamic interaction between Src and C-terminal Src kinase in integrin alphaIIbbeta3-mediated signaling to the cytoskeleton.
Integrin-bound Src tyrosine kinase mediates alpha(IIb)beta(3) out-side-in signaling to the cytoskeleton required for platelet adhesion and thrombus formation. Src activation (signal initiation) by phosphorylation of Tyr-418 occurs at lamellipodia leading edges. However, little is known about Src inactivation mediated by C-terminal Src kinase (Csk) Tyr-529 phosphorylation. In an established platelet model cell line (A5-Chinese hamster ovary), we studied the inactivation of Src during alpha(IIb)beta(3)-mediated adhesion to fibrinogen with live cell fluorescence resonance energy transfer (FRET) microscopy. Imaging revealed highly dynamic Src-Csk interactions at the leading edges of active lamellipodia. The Src-Csk interaction followed a highly dynamic pattern. Every 2-3 min, Src-Csk complexes moved inward in the cell, reorganized, and formed stable focal adhesions. These accumulations were primarily seen during retraction of lamellipodia, whereas no interaction was observed during protrusions. Western blot analysis during the run time of FRET signaling revealed an increase in Csk-mediated SrcTyr-529 phosphorylation with a parallel decline of tyrosine 418 phosphorylation. Mutation analysis provided additional insights into the role of Src. Although inactivation of Csk (CskK222R) had no effect on cell adhesion and spreading efficiency, cells with constitutively active expressed Src (SrcY529F) exhibited hardly any adhesion and no spreading. The few adherent cells showed weak focal adhesions that were disorganized and oversized. The data clearly demonstrate the important role of tight Src control by Csk for functional cell adhesion and spreading. The novel experimental FRET approach reported here for the inactivation of Src can be readily applied to other integrin and signaling pathways, including closely related Src family kinase members.
Vielreicher
Martin
M
Institute of Clinical Biochemistry and Pathobiochemistry, University of Wurzburg, Wurzburg, D-97080, Germany; Molecular Microscopy Group, Rudolf-Virchow-Center, University of Wurzburg, D-97080 Wurzburg, Germany.
Harms
Gregory
G
Molecular Microscopy Group, Rudolf-Virchow-Center, University of Wurzburg, D-97080 Wurzburg, Germany.
Butt
Elke
E
Institute of Clinical Biochemistry and Pathobiochemistry, University of Wurzburg, Wurzburg, D-97080, Germany.
Walter
Ulrich
U
Institute of Clinical Biochemistry and Pathobiochemistry, University of Wurzburg, Wurzburg, D-97080, Germany. Electronic address: uwalter@klin-biochem.uni-wuerzburg.de.
Obergfell
Achim
A
Institute of Clinical Biochemistry and Pathobiochemistry, University of Wurzburg, Wurzburg, D-97080, Germany.
D000818:Animals; D016466:CHO Cells; D000081247:CSK Tyrosine-Protein Kinase; D002448:Cell Adhesion; D006224:Cricetinae; D003412:Cricetulus; D003599:Cytoskeleton / Q000378:metabolism*; D004252:DNA Mutational Analysis; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D010973:Platelet Adhesiveness; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D015398:Signal Transduction; D019061:src-Family Kinases / Q000378:metabolism*
D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D011518:Proto-Oncogene Proteins; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases; C000631346:CSK protein, human