Blood platelets are anucleated cells with unique receptors that respond to blood vessel injury. One of such receptors is integrin alphaIIb-beta3, the fibrinogen receptor. Integrin alphaIIb-beta3, which is in a low-affinity state on resting platelets, changes its conformation upon platelet activation by an agonist and binds fibrinogen, thus initiating both physiological hemostasis and pathological thrombosis. The affinity of alphaIIb-beta3 to its ligand is modulated by cytoplasmic signals, through a process termed inside-out signaling which involves the propagation of signals from the cytoplasm to the extracellular domain in response to intracellular signaling. The binding of fibrinogen induces a cascade of signals inside the platelet termed outside-in signaling that includes tyrosine phosphorylation, cytoskeletal rearrangement and association of alphaIIb-beta3 with the cytoskeleton. As part of the original project, we have identified CIB (Calcium- and Integrin-Binding) protein that interacts specifically with the cytoplasmic domain of alphaIIb. CIB translocates to the cytoskeleton in an aggregation-dependent manner. CIB, which is present on the membranes of unactivated platelets, accumulates at the filopodia upon platelet activation. CIB associates with integrin alphaIIb-beta3and Plk3, a serine/threonine kinase belonging to the polo-like kinase family. Association of CIB with alphaIIb-beta3 is required for platelet spreading on fibrinogen. CIB overexpression in cells induces multinucleation (polyploidy). The proposed project is aimed at further understanding the role of CIB and Plk3 in the modulation of integrin alphaIIb-beta3 function, which may shed light on the mechanism of regulation of cellular processes such as megakaryocyte differentiation, platelet activation and cell migration.
Specific aims of the project are to 1) Study the mechanism of regulation of integrin alphaIIb-beta3 (GPIIb/IIIa) by CIB; 2) Identify the role of Plk3 activity in platelet function; 3) Determine the role of CIB and Plk3 in megakaryocytopoiesis. We plan to use immunological studies in human platelets, a heterologous expression system in CHO cells, and platelets from CIB-null mice to determine the physiological function of CIB in platelet activation. To study the role of CIB and Plk3 in TPO-induced megakaryocytopoiesis, we will use real-time PCR and western blot analysis. We will also characterize the CIB promoter and use CIB-null mice to determine the role of CIB in this process. Since the integrin alphaIIb-beta3 links platelets to each other and to specific components of the vascular wall, it initiates both physiological hemostasis and pathological thrombosis. In addition, alphaIIb-beta3 has been shown to mediate contraction of fibrin clots. Information on the mechanism of activation of the platelet integrin alphaIIb-beta3 will help in the regulation of these processes in platelets and in other cells. Further, characterization of CIB and its interactions with integrin alphaIIb-beta3 and Plk3 may help in delineating the mechanisms by which cytoplasmic signals modulate integrin function.
|Naik, Meghna U; Stalker, Timothy J; Brass, Lawrence F et al. (2012) JAM-A protects from thrombosis by suppressing integrin ?IIb?3-dependent outside-in signaling in platelets. Blood 119:3352-60|
|Kostyak, John C; Naik, Meghna U; Naik, Ulhas P (2012) Calcium- and integrin-binding protein 1 regulates megakaryocyte ploidy, adhesion, and migration. Blood 119:838-46|
|Naik, Meghna U; Naik, Ulhas P (2011) Contra-regulation of calcium- and integrin-binding protein 1-induced cell migration on fibronectin by PAK1 and MAP kinase signaling. J Cell Biochem 112:3289-99|
|Kostyak, John C; Naik, Ulhas P (2011) Calcium- and integrin-binding protein 1 regulates endomitosis and its interaction with Polo-like kinase 3 is enhanced in endomitotic Dami cells. PLoS One 6:e14513|
|Naik, Meghna U; Pham, Ngoc T; Beebe, Kristin et al. (2011) Calcium-dependent inhibition of polo-like kinase 3 activity by CIB1 in breast cancer cells. Int J Cancer 128:587-96|
|Naik, Meghna U; Naik, Ulhas P (2011) Calcium- and integrin-binding protein 1 regulates microtubule organization and centrosome segregation through polo like kinase 3 during cell cycle progression. Int J Biochem Cell Biol 43:120-9|
|Naik, M U; Nigam, A; Manrai, P et al. (2009) CIB1 deficiency results in impaired thrombosis: the potential role of CIB1 in outside-in signaling through integrin alpha IIb beta 3. J Thromb Haemost 7:1906-14|