Systemic lupus erythematosus (SLE) is characterized by disordered T lymphocyte signal transduction. T cells exhibit impaired protein kinase A (PKA)-catalyzed protein phosphorylation due to a profound deficiency of type I PKA (PKA-I) isozyme phosphotransferase activity. Recently, we identified a concomitant deficiency of PKA-II isozyme activity in SLE T cells. Deficient PKA-II activity is associated with (a) autophosphorylation and aberrant translocation of the beta isoform of the type II regulatory subunit (RIIbeta) from the cytosol to the nucleus; (b) accumulation and retention of nuclear RIIbeta; and, (c) reduced or undetectable c-Fos cytosolic protein. Therefore, we hypothesize that aberrant nuclear translocation of autophosphorylated RIIbeta results in (a) deficient PKA-II activity, (b) under-phosphorylation of nuclear cAMP response element binding protein (CREB) transcription factor, (c) impaired c-fos transcriptional activation, (d) reduced levels of c-fos transcript and c-Fos protein, (e) decreased formation of AP-1 transcription factor, and (f) impaired IL-2 transcriptional activation.
The specific aims of this proposal are: (l) To demonstrate that autophosphorylated RIIbeta-subunit is a transcription factor that forms a RIIb-CREB heteromeric complex and acts as a transcriptional repressor of CREB-mediated c-fos transcription in normal primary T cells; (2) To identify the mechanism(s) leading to aberrant translocation of the RIIbeta-subunit from the cytosol to the nucleus in SLE T cells; (3) To determine if PKA-II-catalyzed phosphorylation of CREB is impaired and hinders its binding to CREB binding protein (CBP) and transcriptional activation of the c-Fos promoter in SLE T cells; and, (4) To determine if there is diminished AP-1 binding to consensus AP-1 sites of the IL-2 promoter/enhancer that results in reduced IL-2 production in SLE T cells. The principal goal of the proposed experiments is to establish the mechanism(s) by which deficient PKA-II isozyme activity contributes to altered c-Fos and IL-2 transcriptional activation, loss of AP-1, and diminished IL-2 production by SLE T cells. Demonstrating a connection between reduced T cell IL-2 production and deficient PKA-II isozyme activity in SLE T cells will address a principal gap in our understanding of the molecular and cellular pathophysiology of T cell immunodysfunctions in SLE.
