Phosphorylation of CLEC-2 is dependent on lipid rafts, actin polymerization, secondary mediators, and Rac.
The C-type lectin-like receptor 2 (CLEC-2) activates platelets through Src and Syk tyrosine kinases via a single cytoplasmic YxxL motif known as a hem immunoreceptor tyrosine-based activation motif (hemITAM). Here, we demonstrate using sucrose gradient ultracentrifugation and methyl-beta-cyclodextrin treatment that CLEC-2 translocates to lipid rafts upon ligand engagement and that translocation is essential for hemITAM phosphorylation and signal initiation. HemITAM phosphorylation, but not translocation, is also critically dependent on actin polymerization, Rac1 activation, and release of ADP and thromboxane A(2) (TxA(2)). The role of ADP and TxA(2) in mediating phosphorylation is dependent on ligand engagement and rac activation but is independent of platelet aggregation. In contrast, tyrosine phosphorylation of the GPVI-FcRgamma-chain ITAM, which has 2 YxxL motifs, is independent of actin polymerization and secondary mediators. These results reveal a unique series of proximal events in CLEC-2 phosphorylation involving actin polymerization, secondary mediators, and Rac activation.
Centre for Cardiovascular Sciences, Institute for Biomedical Research, The College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK. a.y.pollitt@bham.ac.uk
Alice Y
AY
Pollitt
Beata
B
Grygielska
Bertrand
B
Leblond
Laurent
L
Désiré
Johannes A
JA
Eble
Steve P
SP
Watson
D000199:Actins / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000378:metabolism; D020816:Amino Acid Motifs; D001792:Blood Platelets / Q000378:metabolism*; D004789:Enzyme Activation / Q000502:physiology; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D037181:Lectins, C-Type / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000378:metabolism*; D021962:Membrane Microdomains / Q000378:metabolism*; D010766:Phosphorylation / Q000502:physiology; D010974:Platelet Aggregation / Q000502:physiology*; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism; D021381:Protein Transport / Q000502:physiology; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D000072377:Syk Kinase; D013928:Thromboxane A2 / Q000378:metabolism; D020830:rac1 GTP-Binding Protein / Q000378:metabolism; D019061:src-Family Kinases / Q000378:metabolism
D000199:Actins; C104778:CLEC2B protein, human; D047908:Intracellular Signaling Peptides and Proteins; D037181:Lectins, C-Type; D008562:Membrane Glycoproteins; D010980:Platelet Membrane Glycoproteins; C107953:RAC1 protein, human; C098341:platelet membrane glycoprotein VI; D013928:Thromboxane A2; D000244:Adenosine Diphosphate; D011505:Protein-Tyrosine Kinases; C000606896:SYK protein, human; D000072377:Syk Kinase; D019061:src-Family Kinases; D020830:rac1 GTP-Binding Protein