title: Stimulation of MAP-2 kinase activity in T lymphocytes by anti-CD3 or anti-Ti monoclonal antibody is partially dependent on protein kinase C.
abstract: Signaling via the alpha-beta T cell Ag receptor (Ti)-CD3 complex is a complicated event that implicates several protein kinases, most notably protein kinase C (PKC). We have recently identified a serine kinase in T lymphocytes with the following characteristics: molecular mass 43 kDa, in vitro substrate affinity for microtubule associated protein 2 (MAP-2) with a preference for Mn2+ during the catalytic reaction, and elution from DEAE resin over a salt range 100 to 200 mM NaCl. This kinase is activated in a rapidly reversible fashion during ligation of CD3/Ti by a process which involves prior phosphorylation; in vitro exposure of activated 43-kDa MAP-2 kinase (MAP-K) to an immobilized phosphatase abrogated its kinase activity. We now show that a MAP-2K response could also be obtained during treatment with mAb to Ti and the specific PKC agonist, PMA. Although the kinetics of the former response was rapidly reversible, PMA elicited a more prolonged response. The dose responsiveness for PMA was similar to the requirements for PKC activation in intact lymphocytes. Moreover, as with PKC, we found that the CD3-induced MAP-2K response could be further enhanced by using a second layer cross-linking antibody. The specificity of CD3/Ti in the Jurkat cell response is demonstrated by the fact that OKT-11(CD2) and anti-CD4 mAb did not stimulate a MAP-2K response. It was also not possible to elicit a response in a Jurkat cell mutant that lacks surface expression of CD3 and Ti. The specificity of PKC in these events was further explored with the cell permeant diacylglycerol, 1-oleoyl-2-acetylglycerol, and the nonagonist phorbol ester, 4 alpha-phorbol 12,13-didecanoate: whereas the former was an effective inducer of the MAP-2K response, the latter failed to yield any stimulation. Prior exposure of Jurkat cells to 100 mM PMA for 24 h eliminated greater than 60% of the MAP-2K response during anti-CD3 treatment. This response could also be inhibited in dose-dependent fashion by prior treatment of Jurkat cells with the potent PKC inhibitor 1-(5-isoquinolinesulfonyl) 2-methylpiperazine dihydrochloride. Although a Ca2(+)-ionophore failed to synergize with PMA at inducing a MAP-2K response, depletion of extracellular Ca2+ by EGTA abrogated anti-CD3 responsiveness. The events culminating in MAP-2K activation were slightly inhibited in the presence of cholera toxin but not pertussis toxin.(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Department of Medicine, UCLA School of Medicine 90024-1680.
authors.forename: A E
authors.initials: AE
authors.lastname: Nel
authors.forename: C
authors.initials: C
authors.lastname: Hanekom
authors.forename: A
authors.initials: A
authors.lastname: Rheeder
authors.forename: K
authors.initials: K
authors.lastname: Williams
authors.forename: S
authors.initials: S
authors.lastname: Pollack
authors.forename: R
authors.initials: R
authors.lastname: Katz
authors.forename: G E
authors.initials: GE
authors.lastname: Landreth
mesh_terms: D000911:Antibodies, Monoclonal; D000945:Antigens, Differentiation, T-Lymphocyte / Q000502:physiology*; D017252:CD3 Complex; D002118:Calcium / Q000502:physiology; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002460:Cell Line; D002772:Cholera Toxin / Q000494:pharmacology; D004789:Enzyme Activation; D006801:Humans; D066298:In Vitro Techniques; D008970:Molecular Weight; D037342:Pertussis Toxin; D010703:Phorbol Esters / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism*; D011948:Receptors, Antigen, T-Cell / Q000502:physiology*; D015398:Signal Transduction; D013601:T-Lymphocytes / Q000201:enzymology*; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
chemical_list: D000911:Antibodies, Monoclonal; D000945:Antigens, Differentiation, T-Lymphocyte; D017252:CD3 Complex; D010703:Phorbol Esters; D011948:Receptors, Antigen, T-Cell; D010566:Virulence Factors, Bordetella; D002772:Cholera Toxin; D037342:Pertussis Toxin; D011494:Protein Kinases; D011493:Protein Kinase C; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002118:Calcium