Immunological identification of protein kinase C-α and protein kinase C-δ in cultured rat mesangial cells: Diffferential sensitivity of the two isoforms towards the protein kinase inhibitor H 7

Abstract

Rat mesangial cells contain both calcium-dependent protein kinase C (PKC) activity, which phosphorylates histone H 1 and endogenous proteins, and calcium-independent, phospholipid-dependent PKC activity, which phosphorylates only endogenous proteins. The calcium-dependent PKC was identified as PKC α by immunoblot analysis and hydroxyapatite chromatography (HPLC). The calcium-insensitive, phospholipid-dependent isoform was identified as PKC δ using similar techniques. The inhibition of the two PKC isoforms by the protein kinase inhibitor H 7 [1-(iso-quinolinyl sulphonyl)-2-methyl piperazine] was examined using both histone H 1 and endogenous proteins as substrates. Phosphorylations catalysed by the calcium-dependent PKC isomform α were almost 90% inhibited when histone H 1 was used, and only 55% when endogenous protein were the substrate. In contrast, the phosphorylation of endogenous proteins catalysed by the calcium-insensitive, phopholipid-dependent PKC δ was not significantly affected by the inhibitor.