Analysis of post-translational modification by electrophoresis—New developments on the functional analysis of retinoylated proteins

Abstract

Post translational protein modification is recognized as an important mechanism for diversification of protein function. Research into signal transduction mechanisms focusing on regulation of transcriptional expression of genes and on the transport or degradation of proteins, is an intensive area of investigation. Covalent modification of opsin by retinal is a critical mechanism by which vitamin A participates in the process of vision. Retinoic acid (RA) is an oxidized form of retinal that is a potent inducer of HL60 cell differentiation, which can achieve complete remission in patients with acute promyelocytic leukemia. While one mechanism by which RA achieves its effects involves RA nuclear receptors, retinoylation (post-translational modification of proteins by RA) represents an alternate non-genomic mechanism. One metabolic pathway for retinoylation involves the intermediate formation of retinoyl-CoA and the transfer and covalent binding of the retinoyl moiety to protein. The regulatory subunit of cAMP-dependent protein kinase (proteinkinase A, PKA)(RIIα) is retinoylated at early time after RA treatment to HL60 cells. Once retinoylated, RIIα units are translocated to the nucleus where phosphorylation of nuclear proteins occurs. This promotes the phosphorylation of nuclear proteins by PKA and this may induce differentiation into granulocyte-like cells. We are currently identifying nuclear proteins phosphorylated by PKA, which are increased by RA treatment and determining the sites on RIIα that are modified. These efforts may clarify actions of RA that function in cooperation with the RA nuclear receptor and retinoylation.