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 Cgamma2 (PLCgamma2). 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 PLCgamma2 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 PLCgamma2. In contrast to the results with CRP, the G protein-coupled receptor agonist thrombin stimulates PI 3-kinase-independent translocation of Btk but not PLCgamma2. In conclusion, these results demonstrate that in mouse megakaryocytes, CRP leads to PI 3-kinase-dependent translocation of PLCgamma2 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.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Agammaglobulinaemia Tyrosine Kinase
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Agammaglobulinemia / enzymology
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Androstadienes / pharmacology
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Animals
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Blood Platelets / drug effects
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Blood Platelets / enzymology
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Calcium / metabolism
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Carrier Proteins*
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Cell Membrane / drug effects
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Cell Membrane / metabolism
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Cells, Cultured
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Enzyme Inhibitors / pharmacology
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Enzyme Precursors / metabolism
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Humans
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Intracellular Signaling Peptides and Proteins
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Isoenzymes / chemistry
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Isoenzymes / metabolism*
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Megakaryocytes / drug effects
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Megakaryocytes / enzymology*
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Mice
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Mice, Mutant Strains
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Models, Biological
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Phosphatidylinositol 3-Kinases / physiology*
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Phosphatidylinositol Phosphates / metabolism
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Phosphoinositide-3 Kinase Inhibitors
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Phospholipase C gamma
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Protein Transport
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Protein-Tyrosine Kinases / metabolism*
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Proteins / pharmacology
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Syk Kinase
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Thrombin / pharmacology
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Type C Phospholipases / chemistry
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Type C Phospholipases / metabolism*
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Wortmannin
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X Chromosome
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src Homology Domains
Substances
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Androstadienes
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Carrier Proteins
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Enzyme Inhibitors
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Enzyme Precursors
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Intracellular Signaling Peptides and Proteins
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Isoenzymes
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Phosphatidylinositol Phosphates
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Phosphoinositide-3 Kinase Inhibitors
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Proteins
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c1q-binding proteins
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phosphatidylinositol 3,4,5-triphosphate
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Protein-Tyrosine Kinases
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Agammaglobulinaemia Tyrosine Kinase
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BTK protein, human
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Btk protein, mouse
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SYK protein, human
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Syk Kinase
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Syk protein, mouse
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Type C Phospholipases
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Phospholipase C gamma
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Thrombin
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Calcium
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Wortmannin