Deficient type I protein kinase A isozyme activity in systemic lupus erythematosus T lymphocytes: II. Abnormal isozyme kinetics.
Systemic lupus erythematosus (SLE) T cells exhibit deficient type I protein kinase A (PKA-1) isozyme phosphotransferase activity, resulting in impaired phosphorylation of plasma membrane-associated proteins. To determine the mechanism of this isozyme deficiency, we studied 16 SLE subjects with a mean (+/-1 SD) SLE disease activity index of 16.7 +/- 8.8 and 16 normal controls. Immunoblotting of type I regulatory (RI) subunit protein in SLE and control T cells demonstrated no significant differences in the amount of protein. Analysis of isozyme kinetics in SLE T cells demonstrated a 2.2-fold increase in the Michaelis-Menten constant, a 2.5-fold increase in the apparent association constant for cAMP, a 3.8-fold decrease in the maximal velocity, and a reduction in the mean maximal binding of cAMP to the RI subunit compared with control T cells. Reduction of the Hill coefficient from 1.2 in normal T cells to 0.7 in SLE T cells indicated a loss of positive cooperativity between cAMP binding sites A and B. An increase in the apparent association constant for cAMP signifies relative resistance to cAMP, indicating that higher intracellular concentrations of cAMP are necessary to activate the isozyme. Because the R subunit of PKA is the only intracellular receptor for cAMP, the abnormal isozyme kinetics may account for the deficiency of PKA-I phosphotransferase activity and impaired PKA-I-catalyzed protein phosphorylation observed in SLE T cells. This disordered isozyme function may contribute to the altered signal transduction and observed cellular immune dysfunctions in SLE. Moreover, these altered isozyme kinetics raise the possibility of a structural defect(s) in the RI subunit.
Kammer
G M
GM
Department of Internal Medicine, Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, NC 27157, USA.
Khan
I U
IU
Kammer
J A
JA
Olorenshaw
I
I
Mathis
D
D
D000328:Adult; D000368:Aged; D002845:Chromatography; D000242:Cyclic AMP / Q000502:physiology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000172:deficiency* / Q000378:metabolism; D004789:Enzyme Activation; D005260:Female; D006801:Humans; D015151:Immunoblotting; D007527:Isoenzymes / Q000172:deficiency* / Q000378:metabolism; D007700:Kinetics; D008180:Lupus Erythematosus, Systemic / Q000201:enzymology* / Q000276:immunology*; D008297:Male; D008875:Middle Aged; D013601:T-Lymphocytes / Q000201:enzymology* / Q000276:immunology*
D007527:Isoenzymes; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases