Activation of resting peripheral blood lymphocytes through the T cell receptor induces rapid phosphorylation of Op18.
Op18 is a highly conserved major cytosolic phosphoprotein that has been implicated in signal transduction in a wide variety of cell types. Freshly isolated peripheral blood lymphocytes (PBL) constitutively express low levels of mostly unphosphorylated Op18. After mitogenic stimulation of PBL, Op18 synthesis is induced at a time when cells are entering S-phase. In this study, we have examined the phosphorylation of Op18 in freshly isolated PBL after activation of the T cell receptor by OKT3. Quantitative analysis of Op18 phosphorylation was undertaken by metabolic labeling with 32Pi and PhosphorImager analysis of two-dimensional gels. After 10 or 15 min of activation by OKT3, one of the three major phosphorylated forms of Op18, designated Op18c, increased approximately 10-fold, which represented a most pronounced change among a large number of phosphoproteins analyzed. In time course experiments, increased Op18 phosphorylation to yield Op18c was observed as early as 2 min. Continued OKT3-induced activation for 20 to 72 h resulted in a further increase in phosphorylated Op18 forms, which paralleled new Op18 synthesis and occurred at a time when cells were entering S-phase, as determined by [3H]-thymidine incorporation. Inhibitors of lymphoid proliferation, cyclosporin A and RPM, had no effect on early (less than 15 min) phosphorylation. Addition of calphostin C, a specific inhibitor of protein kinase C, 1 min prior to stimulation of resting T cells with OKT3 completely inhibited further phosphorylation of Op18. Incubation of PBL with calphostin C for 75 min decreased constitutive levels of phosphorylated Op18. In contrast, inhibition of cyclic nucleotide-dependent protein kinases with HA1004 had no effect on Op18 phosphorylation. Activation of cAMP-dependent protein kinase with Forskolin or 8Br-cAMP did not increase Op18 phosphorylation. Our results suggest that Op18 phosphorylation is mediated by protein kinase C activation as an early event in T cell activation through the T cell receptor.
Strahler
J R
JR
University of Michigan Medical School, Department of Pediatrics, Ann Arbor 48109.
Hailat
N
N
Lamb
B J
BJ
Rogers
K P
KP
Underhill
J A
JA
Melhem
R F
RF
Keim
D R
DR
Zhu
X
X
Kuick
R D
RD
Fox
D A
DA
D002478:Cells, Cultured; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D066298:In Vitro Techniques; D008213:Lymphocyte Activation; D008868:Microtubule Proteins; D016853:Muromonab-CD3 / Q000276:immunology; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D011494:Protein Kinases / Q000378:metabolism; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism*; D015398:Signal Transduction; D050777:Stathmin; D013997:Time Factors
D008868:Microtubule Proteins; D016853:Muromonab-CD3; D010750:Phosphoproteins; D011948:Receptors, Antigen, T-Cell; C493995:STMN1 protein, human; D050777:Stathmin; D011494:Protein Kinases; D011493:Protein Kinase C