Src kinase and PI 3-kinase as a transduction pathway in ceramide-induced contraction of colonic smooth muscle.
Ceramide mediates sustained contraction of smooth muscle cells. C2 ceramide induced a rapid increase in Src kinase activity within 15 s, peaked at 1 min, and was sustained up to 8 min. Contraction and Src kinase activity were inhibited in cells incubated in Ca2+-free medium containing 2 mM EGTA and in cells preincubated with herbimycin A, a Src kinase inhibitor. Immunoblotting using a phosphospecific anti-Src (416Y) antibody showed a ceramide-induced increase in pp60(src) tyrosine phosphorylation. Immunoprecipitation using an anti-phosphotyrosine antibody followed by Western immunoblotting using a monoclonal IgG anti-phosphoinositide 3-kinase NH2 terminal-SH2 domain antibody showed a ceramide-induced increase in phosphoinositide 3-kinase (PI 3-kinase) tyrosine phosphorylation at a protein mass corresponding to 85 kDa, the regulatory subunit of PI 3-kinase, which contains the Src kinase binding site. PI 3-kinase phosphorylation was inhibited by herbimycin A and by the PI 3-kinase inhibitors wortmannin and LY-294002. Preincubation of cells with herbimycin A or PI 3-kinase inhibitors also resulted in an inhibition of mitogen-activated protein (MAP) kinase p42 and p44 activities as seen on Western blots. In summary, we found that 1) the maintenance of sustained contraction is dependent on extracellular Ca2+; 2) ceramide activates a nonreceptor tyrosine kinase pathway through activation of pp60(src) and PI 3-kinase; and 3) the converging signals are probably through activation of MAP kinase.
Ibitayo
A I
AI
Department of Pediatrics, University of Michigan Medical Center, Ann Arbor, Michigan 48109-0656, USA.
Tsunoda
Y
Y
Nozu
F
F
Owyang
C
C
Bitar
K N
KN
D000595:Amino Acid Sequence; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D016227:Benzoquinones; D002118:Calcium / Q000494:pharmacology; D002867:Chromones / Q000494:pharmacology; D003106:Colon / Q000187:drug effects / Q000502:physiology*; D004533:Egtazic Acid / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D066298:In Vitro Techniques; D007700:Kinetics; D047029:Lactams, Macrocyclic; D008954:Models, Biological; D008969:Molecular Sequence Data; D009025:Morpholines / Q000494:pharmacology; D009119:Muscle Contraction / Q000187:drug effects / Q000502:physiology*; D009130:Muscle, Smooth / Q000187:drug effects / Q000502:physiology*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000378:metabolism; D011809:Quinones / Q000494:pharmacology; D011817:Rabbits; D017828:Rifabutin / Q000031:analogs & derivatives; D015398:Signal Transduction; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D013379:Substrate Specificity; D000077191:Wortmannin; D018909:src Homology Domains
D000730:Androstadienes; D016227:Benzoquinones; D002867:Chromones; D004791:Enzyme Inhibitors; D047029:Lactams, Macrocyclic; D009025:Morpholines; C064769:N-acetylsphingosine; D011809:Quinones; D017828:Rifabutin; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D004533:Egtazic Acid; C020754:herbimycin; D019869:Phosphatidylinositol 3-Kinases; D011505:Protein-Tyrosine Kinases; D016392:Proto-Oncogene Proteins pp60(c-src); D013110:Sphingosine; D002118:Calcium; D000077191:Wortmannin