Phosphatidylinositol 3-kinase–dependent translocation of phospholipase Cγ2 in mouse megakaryocytes is independent of Bruton tyrosine kinase translocation

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Abstract
Activation of the collagen receptor glycoprotein VI (GPVI) by a collagen-related peptide (CRP) induces stimulation of platelets and megakaryocytes through the phosphatidylinositol (PI) 3-kinase–dependent pathway leading to activation of Bruton tyrosine kinase (Btk) and phospholipase Cγ2 (PLCγ2). Here, we present evidence that both proteins undergo PI 3-kinase–dependent translocation to the plasma membrane on CRP stimulation that is markedly inhibited by wortmannin and LY294002. Translocation of PLCγ2 but not Btk is also seen in megakaryocytes from X-linked immunodeficiency mice, which have a mutation that reduces the affinity of the pleckstrin homology (PH) domain of Btk for PI 3,4,5-trisphosphate (PI 3,4,5-P3). Activation of PC12 cells by epidermal growth factor (EGF) results in increased PI 3-kinase activity and high PI 3,4,5-P3 levels that trigger translocation of the green fluorescent protein (GFP)–labeled PH of Btk, but not the GFP-labeled PH and tandem Src homology 2 (SH2) domains of PLCγ2. In contrast to the results with CRP, the G protein–coupled receptor agonist thrombin stimulates PI 3-kinase–independent translocation of Btk but not PLCγ2. In conclusion, these results demonstrate that in mouse megakaryocytes, CRP leads to PI 3-kinase–dependent translocation of PLCγ2 and Btk that are independent of one another, whereas thrombin only induces translocation of Btk through a pathway that is independent of PI 3-kinase activity.

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