Inhibition of Polo-Like Kinase 3 by CIB1 Facilitates Outside-In Signaling through αIIbβ3 in Platelets.

Integrin α_IIbβ₃, the platelet fibrinogen receptor, is involved in bi-directional signaling during platelet activation. It has been shown that the β₃ subunit of this receptor is phosphorylated on both threonine and tyrosine. Although, the role of tyrosine phosphorylation is well studied, the role of threonine phosphorylation is not well understood. Here we identify the kinase responsible for threonine phosphoruylation of β₃ and provide a mechanistic significance of this phosphorylation in outside-in signaling. We found that calcium- and integrin-binding protein 1 (CIB1), which specifically interacts with the cytoplasmic domain of α_IIb, is not involved in inside-out signaling, but regulates outside-in signaling through α_IIbβ₃. CIB1 is known to interact with and regulate several serine/threonine protein kinases. Among these, p21 activated kinase 1 (PAK-1) is highly expressed in platelets. We found that CIB1 does not colocalize with PAK-1 during outside in signaling induced by adhesion of platelets to immobilized fibrinogen. When searched for the expression of other kinases that are known to interact with CIB1, we found that polo-like kinase 3 (Plk3) is expressed in platelets and is responsible for the constitutive phosphorylation of β₃. Plk3 is constitutively active in resting platelets but is rendered inactive during outside in signaling as a result of CIB1 binding in a calcium dependent manner. Plk3 becomes colocalized with CIB1 at the filopodia when platelets change shape in response to an agonist and remain associated with both CIB1 and α_IIb as platelets spread on immobilized fibrinogen, as indicated by immunoprecipitation experiments. These results suggest that in resting platelets, constitutively active Plk3 maintains β₃ in a threonine phosphorylated state which is nonpermissive for activation. Upon platelet activation by agonist CIB1 associates with Plk3 and renders it inactive in a calcium-dependent manner. This allows serine/threonine phosphatases, such as PP1-c to dephosphorylate β₃ on threonine and facilitate outside-in signaling. Thus we have identified the kinase responsible for threonine phosphorylation of β₃ and have provided a possible mechanism of the regulation of outside-in signaling.