A p85 subunit-independent p110alpha PI 3-kinase colocalizes with p70 S6 kinase on actin stress fibers and regulates thrombin-stimulated stress fiber formation in swiss 3T3 cells.
The signaling pathways linking receptor activation to actin stress fiber rearrangements during growth factor-induced cell shape change are still to be determined. Recently our laboratory demonstrated the involvement of p70 S6 kinase (p70(s6k)) activation in thrombin-induced stress fiber formation in Swiss 3T3 cells. The present work shows that thrombin-induced p70(s6k) activation is inhibited by the PI 3-kinase inhibitors wortmannin and LY-294002. These inhibitors also significantly reduced thrombin-induced stress fiber formation, demonstrating a role for PI 3-kinase activity in this process, most likely upstream of p70(s6k). Furthermore, the p110alpha form of PI 3-kinase was localized to actin stress fibers, as was previously shown for p70(s6k), as well as to a golgi-like distribution. In contrast, PI 3-kinase p110gamma colocalized with microtubules. The PI 3-kinase p85 subunit, known to be capable of association with p110alpha, was present in a predominantly golgi-like distribution with no presence on actin filaments, suggesting the existence of distinctly localized PI 3-kinase pools. Immunodepletion of p85 from cell lysates resulted in only partial depletion of p110alpha and p110alpha-associated PI 3-kinase activity, confirming the presence of a p85-free p110alpha pool located on the actin stress fibers. Our data, therefore, point to the importance of subcellular localization of PI 3-kinase in signal transduction and to a novel action of p85 subunit-independent PI 3-kinase p110alpha in the stimulation by thrombin of p70(s6k) activation and actin stress fiber formation.
Johanson
S O
SO
John Curtin School of Medical Research, Australian National University, Canberra, 2600, Australia.
Naccache
P A
PA
Crouch
M F
MF
D016475:3T3 Cells; D000199:Actins; D000595:Amino Acid Sequence; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D002867:Chromones / Q000494:pharmacology; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004815:Epidermal Growth Factor / Q000494:pharmacology; D019084:Fluorescent Antibody Technique, Indirect; D051379:Mice; D008969:Molecular Sequence Data; D009025:Morpholines / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011817:Rabbits; D019893:Ribosomal Protein S6 Kinases / Q000378:metabolism*; D013917:Thrombin / Q000494:pharmacology; D000077191:Wortmannin
D000199:Actins; D000730:Androstadienes; D002867:Chromones; D004791:Enzyme Inhibitors; D004815:Epidermal Growth Factor; D009025:Morpholines; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D019893:Ribosomal Protein S6 Kinases; D013917:Thrombin; D000077191:Wortmannin; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one