title: Immunoreceptor tyrosine-based inhibitory motif (ITIM)-mediated inhibitory signaling is regulated by sequential phosphorylation mediated by distinct nonreceptor tyrosine kinases: a case study involving PECAM-1.
abstract: The activation state of many blood and vascular cells is tightly controlled by a delicate balance between receptors that contain immunoreceptor tyrosine-based activation motifs (ITAMs) and those that contain immunoreceptor tyrosine-based inhibitory motifs (ITIMs). Precisely how the timing of cellular activation by ITAM-coupled receptors is regulated by ITIM-containing receptors is, however, poorly understood. Using platelet endothelial cell adhesion molecule 1 (PECAM-1) as a prototypical ITIM-bearing receptor, we demonstrate that initiation of inhibitory signaling occurs via a novel, sequential process in which Src family kinases phosphorylate the C-terminal ITIM, thereby enabling phosphorylation of the N-terminal ITIM of PECAM-1 by other Src homology 2 domain-containing nonreceptor tyrosine kinases (NRTKs). NRTKs capable of mediating the second phosphorylation event include C-terminal Src kinase (Csk) and Bruton's tyrosine kinase (Btk). Btk and Csk function downstream of phosphatidylinositol 3-kinase (PI3K) activation during ITAM-dependent platelet activation. In ITAM-activated platelets that were treated with a PI3K inhibitor, PECAM-1 was phosphorylated but did not bind the tandem SH2 domain-containing tyrosine phosphatase SHP-2, indicating that it was not phosphorylated on its N-terminal ITIM. Csk bound to and phosphorylated PECAM-1 more efficiently than did Btk and required its SH2 domain to perform these functions. Additionally, the phosphorylation of the N-terminal ITIM of Siglec-9 by Csk is enhanced by the prior phosphorylation of its C-terminal ITIM, providing evidence that the ITIMs of other dual ITIM-containing receptors are also sequentially phosphorylated. On the basis of these findings, we propose that sequential ITIM phosphorylation provides a general mechanism for precise temporal control over the recruitment and activation of tandem SH2 domain-containing tyrosine phosphatases that dampen ITAM-dependent signals.
authors: Blood Research Institute, BloodCenter of Wisconsin, Milwaukee, Wisconsin 53226, USA.
authors: Benjamin E
authors: BE
authors: Tourdot
authors: Michelle K
authors: MK
authors: Brenner
authors: Kathleen C
authors: KC
authors: Keough
authors: Trudy
authors: T
authors: Holyst
authors: Peter J
authors: PJ
authors: Newman
authors: Debra K
authors: DK
authors: Newman
mesh_terms: D000077329:Agammaglobulinaemia Tyrosine Kinase; D020816:Amino Acid Motifs; D001792:Blood Platelets / Q000378:metabolism*; D000081247:CSK Tyrosine-Protein Kinase; D003593:Cytoplasm / Q000378:metabolism; D006801:Humans; D010748:Phosphopeptides / Q000378:metabolism; D010766:Phosphorylation; D019408:Platelet Endothelial Cell Adhesion Molecule-1 / Q000378:metabolism*; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D015398:Signal Transduction; D018909:src Homology Domains; D019061:src-Family Kinases / Q000378:metabolism
chemical_list: D010748:Phosphopeptides; D019408:Platelet Endothelial Cell Adhesion Molecule-1; D010980:Platelet Membrane Glycoproteins; C098341:platelet membrane glycoprotein VI; D011505:Protein-Tyrosine Kinases; D000077329:Agammaglobulinaemia Tyrosine Kinase; C000625949:BTK protein, human; D000081247:CSK Tyrosine-Protein Kinase; C060902:lyn protein-tyrosine kinase; D019061:src-Family Kinases; C000631346:CSK protein, human