Phosphorylation sites in the integrin beta3 cytoplasmic domain in intact platelets.
Protein seryl/threonyl phosphatase inhibitors such as calyculin A block inside-out and outside-in platelet signaling. Our studies demonstrate that the addition of calyculin A blocks platelet adhesion and spreading on fibrinogen, responses that depend on integrin alphaIIb beta3 signaling. We hypothesized that this reflects a change in alphaIIb beta3 structure caused by a specific state of phosphorylation. We show that addition of calyculin A leads to increased phosphorylation of the beta3 subunit, and phosphoamino acid analysis reveals that only threonine residues become phosphorylated; sequence analysis by Edman degradation established that threonine 753 became stoichiometrically phosphorylated during inhibition of platelet phosphatases by calyculin A. This region of beta3 is linked to outside-in signaling such as platelet spreading responses. The effect of calyculin A on platelet adhesion and spreading and on the phosphorylation of T-753 in beta3 is reversed by the calcium ionophore A23187, demonstrating that these effects of calyculin A are not generally toxic ones. We propose that phosphorylation of beta3 on threonine 753, a region of beta3 linked to outside-in signaling, may be a mechanism by which integrin alphaIIb beta3 function is regulated.
Lerea
K M
KM
Department of Cell Biology and Anatomy, New York Medical College, Valhalla, New York 10595, USA. ken_lerea@nymc.edu
Cordero
K P
KP
Sakariassen
K S
KS
Kirk
R I
RI
Fried
V A
VA
D001792:Blood Platelets / Q000166:cytology / Q000378:metabolism*; D002448:Cell Adhesion / Q000187:drug effects; D003593:Cytoplasm / Q000378:metabolism*; D006801:Humans; D008387:Marine Toxins; D010080:Oxazoles / Q000494:pharmacology; D010766:Phosphorylation; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000737:chemistry / Q000378:metabolism*; D015398:Signal Transduction; D013912:Threonine / Q000378:metabolism
D008387:Marine Toxins; D010080:Oxazoles; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D013912:Threonine; C059041:calyculin A