Phosphorylation of T-lymphocyte plasma membrane-associated proteins by ectoprotein kinases: implications for a possible role for ectophosphorylation in T-cell effector functions.
Extracellular adenosine triphosphate (ATPo) has been suggested to play a role in lymphocyte effector functions. Recently, it has been suggested that MgATP2- may be the molecular species which is involved in modulating the lytic interaction between cytotoxic T-lymphocytes (CTL) and their target cells. In this study, we provide evidence that ATPo mediates the phosphorylation of extracellular proteins on T-lymphocytes through the action of ectoprotein kinases. The ectophosphorylation is temperature-dependent, supported by Mg2+ and Mn2+, and both ATP and GTP, whereas kinase activity and/or substrates were removed by pretreatment of intact lymphocytes with trypsin. We show the presence of extracellular ATP/GTP-binding sites, indicating the presence of ectoenzymes on intact lymphocytes. The major ectoprotein kinase was identified as a casein kinase II-like protein kinase and could be inhibited by heparin, whereas its activity was enhanced by spermine. The ectoprotein kinase showed remarkable substrate specificity, phosphorylating the serum protein vitronectin, but not fibronectin. In experiments with the cell-impermeable protein kinase inhibitor K-252b, we demonstrate the possible functional importance of ectoprotein kinase in CTL-mediated cytotoxicity, i.e., target cell death was completely blocked by K-252b without affecting intracellular phosphorylation. These results suggest that ectoprotein phosphorylation may possibly be an important event in immunologically relevant cell-cell interactions.
Redegeld
F A
FA
Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
Smith
P
P
Apasov
S
S
Sitkovsky
M V
MV
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002227:Carbazoles / Q000494:pharmacology; D047390:Casein Kinase II; D002412:Cations / Q000494:pharmacology; D002462:Cell Membrane / Q000201:enzymology*; D003601:Cytotoxicity Tests, Immunologic; D003602:Cytotoxicity, Immunologic / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000378:metabolism; D026121:Indole Alkaloids; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000187:drug effects / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases; D013379:Substrate Specificity; D013602:T-Lymphocytes, Cytotoxic / Q000201:enzymology*
D002227:Carbazoles; D002412:Cations; D004791:Enzyme Inhibitors; D026121:Indole Alkaloids; D008565:Membrane Proteins; D047428:Protein Kinase Inhibitors; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; C049985:staurosporine aglycone; D011494:Protein Kinases; C034454:ectoprotein kinase; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases