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 non-receptor 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.

Distinct roles of immunoreceptor tyrosine‐based motifs in immunosuppressive indoleamine 2,3‐dioxygenase 1

“…In a similar fashion, the prototypical ITIM‐bearing receptor FcγRIIB contains two distinct ITIMs that can recruit different molecular partners, such as SHIP phosphatases and the adapters Grb2 and Grap 38. A mechanism of phosphorylation of dual ITIM‐containing receptors has been proposed for Siglec‐9 and PECAM‐1, involving a sequential phosphorylation of the two ITIM motifs operated by different Src tyrosine kinases 39. Several examples of dual/multiple ITIM‐containing receptors exist where consecutive phosphorylation occurs for generating sequential docking sites anchoring different intermediates that convey in a unique outcome, including the transduction of the inhibitory signaling.…”

The BCR-ABL inhibitor ponatinib inhibits platelet immunoreceptor tyrosine-based activation motif (ITAM) signaling, platelet activation and aggregate formation under shear

“…In this study, we find that, in vitro, while treatment of platelets with imatinib or nilotinib had only marginal effects on platelet tyrosine kinase phosphorylation, ponatinib markedly reduced the overall levels of tyrosine phosphorylated proteins and dramatically reduced the levels of activated Src and phosphorylated Lyn in platelets activated on fibrinogen or collagen surfaces. Phosphorylation of the Bruton’s tyrosine kinase (BTK) and LAT, which occur downstream of Src activation in platelet GPVI signaling [15, 23, 30], was also inhibited by treatment with ponatinib.…”

“…At the onset of the GPVI/ITAM platelet signaling processes, an interplay amongst platelet SFKs and other tyrosine kinases and phosphatases positively and negatively orchestrates the temporality of platelet activation [15, 30]. Such signaling events are complex, as SFKs such as Lyn can both inhibit or potentiate aspects of platelet function, including platelet spreading, aggregation and dense granule secretion [21, 31, 32].…”

Endothelial functions of platelet/endothelial cell adhesion molecule-1 (CD31)

“…Though largely unstructured, the cytoplasmic domain contains a single lipid-associated segment that is susceptible, upon cellular activation, to inducible, sequential phosphorylation [18,33]; first of serine residues that release a membrane-associated control region from the inner face of the plasma membrane (see Figure 1), and then of tyrosines 663 and 686, each of which exist within immunoreceptor tyrosine-based inhibitory motifs (ITIMs). PECAM-1 ITIMs, when phosphorylated, recruit the protein-tyrosine phosphatase, SHP-2 [34], resulting in formation of a PECAM-1/SHP-2 complex that functions in circulating blood cells to inhibit a plethora of tyrosine kinase-initiated cellular activation events [35].…”