SENDAI VIRUS P-PROTEIN IS CONSTITUTIVELY PHOSPHORYLATED AT SERINE249 - HIGH PHOSPHORYLATION POTENTIAL OF THE P-PROTEIN

Previously we showed that the Sendai virus P protein (568 aa) in virus -infected cells and in virions was primarily and constitutively phosph orylated on serine(s) in a single tryptic phosphopeptide TP1. By two-d imensional thin-layer electrophoresis and chromatography analysis of t ryptic phosphopeptides of several deletion and point mutants of the P protein, we now show that the sole phosphorylation site in TP1 is seri ne249. Interestingly, when serine249 was deleted or mutagenized altern ate potential serine sites were more heavily phosphorylated. A similar effect was observed when the deletion was very close to serine249 (De lta 208-236). Mutagenesis of proline250 to alanine abrogated phosphory lation at serine249 suggesting that proline250 is essential for the pr imary phosphorylation of the P protein. Conceivably, serine249 phospho rylation is mediated by a proline-directed protein kinase. This findin g is unusual because a majority of the P proteins from other negative- strand RNA viruses have been shown to be phosphorylated primarily by c asein kinase II. Our results demonstrate that the P protein has a stro ng potency to remain phosphorylated. Based on our previous and present results, we suggest that the phosphorylation sites on P are dependent on the accessibility of phosphatases rather than kinases as all poten tial sites are about equally competent for phosphorylation. We propose that phosphorylation is important for maintaining the structural inte grity of the Sendai virus P protein. (C) 1996 Academic Press, Inc.