Interleukin-1 and synovial protein kinase C: Identification of a novel, 35 kDa cytosolic substrate

We have been examining the role of protein kinase C (PKC) in synovial cell activation in response to interleukin-1 (IL-1). Attempts to measure PKC in soluble extracts of synovial fibroblasts by standard techniques failed. Western blotting with anti-PKC antibodies detected only a low level of PKC in synovial cells compared to rat basophilic leukemia cells and crude brain extracts. However, synovial PKC could be detected by measuring the Ca²⁺-and phospholipid-dependent phosphorylation of endogenous substrates. In this way, a 35 kDa protein was identified as the major endogenous cytosolic substrate for PKC. Treatment of synoviocytes with phorbol myristate acetate (PMA) strongly induced the synthesis of neutral metalloproteinases (NPs) and prostaglandin E₂ (PGE₂). Both Western blotting and assays based upon phosphorylation of the 35 kDa protein confirmed translocation of PKC from the cytosol in response to PMA. Although IL-1 induced the NPs and PGE₂, it did so without detectable translocation of PKC. There thus appear to be PKC-dependent and PKC-independent routes of synovial cell activation. Our data suggest that IL-1 uses the latter.