The CC chemokine monocyte chemotactic peptide-1 activates both the class I p85/p110 phosphatidylinositol 3-kinase and the class II PI3K-C2alpha.
The cellular effects of MCP-1 are mediated primarily by binding to CC chemokine receptor-2. We report here that MCP-1 stimulates the formation of the lipid products of phosphatidylinositol (PI) 3-kinase, namely phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate (PI 3,4,5-P3) in THP-1 cells that can be inhibited by pertussis toxin but not wortmannin. MCP-1 also stimulates an increase in the in vitro lipid kinase activity present in immunoprecipitates of the class 1A p85/p110 heterodimeric PI 3-kinase, although the kinetics of activation were much slower than observed for the accumulation of PI 3,4,5-P3. In addition, this in vitro lipid kinase activity was inhibited by wortmannin (IC50 = 4.47 +/- 1.88 nM, n = 4), and comparable concentrations of wortmannin also inhibited MCP-stimulated chemotaxis of THP-1 cells (IC50 = 11.8 +/- 4.2 nM, n = 4), indicating that p85/p110 PI 3-kinase activity is functionally relevant. MCP-1 also induced tyrosine phosphorylation of three proteins in these cells, and a fourth tyrosine-phosphorylated protein co-precipitates with the p85 subunit upon MCP-1 stimulation. In addition, MCP-1 stimulated lipid kinase activity present in immunoprecipitates of a class II PI 3-kinase (PI3K-C2alpha) with kinetics that closely resembled the accumulation of PI 3,4,5-P3. Moreover, this MCP-1-induced increase in PI3K-C2alpha activity was insensitive to wortmannin but was inhibited by pertussis toxin pretreatment. Since this mirrored the effects of these inhibitors on MCP-1-stimulated increases in D-3 phosphatidylinositol lipid accumulation in vivo, these results suggest that activation of PI3K-C2alpha rather than the p85/p110 heterodimer is responsible for mediating the in vivo formation of D-3 phosphatidylinositol lipids. These data demonstrate that MCP-1 stimulates protein tyrosine kinases as well as at least two separate PI 3-kinase isoforms, namely the p85/p110 PI 3-kinase and PI3K-C2alpha. This is the first demonstration that MCP-1 can stimulate PI 3-kinase activation and is also the first indication of an agonist-induced activation of the PI3K-C2alpha enzyme. These two events may play important roles in MCP-1-stimulated signal transduction and biological consequences.
Turner
S J
SJ
Pharmacology Group, Department of Pharmacy and Pharmacology, Bath University, Claverton Down, Bath, Avon BA2 7AY, United Kingdom.
Domin
J
J
Waterfield
M D
MD
Ward
S G
SG
Westwick
J
J
D000730:Androstadienes / Q000494:pharmacology; D002460:Cell Line; D018932:Chemokine CCL2 / Q000494:pharmacology*; D002633:Chemotaxis / Q000502:physiology; D004789:Enzyme Activation / Q000187:drug effects*; D006801:Humans; D007700:Kinetics; D037342:Pertussis Toxin; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D019000:Phosphotyrosine / Q000032:analysis; D011233:Precipitin Tests; D015398:Signal Transduction / Q000502:physiology; D010566:Virulence Factors, Bordetella / Q000494:pharmacology; D000077191:Wortmannin
D000730:Androstadienes; D018932:Chemokine CCL2; D018129:Phosphatidylinositol Phosphates; D010750:Phosphoproteins; D010566:Virulence Factors, Bordetella; C060974:phosphatidylinositol 3,4,5-triphosphate; C060980:phosphatidylinositol 3,4-diphosphate; D019000:Phosphotyrosine; D037342:Pertussis Toxin; D019869:Phosphatidylinositol 3-Kinases; D000077191:Wortmannin