REGULATION OF IN-VITRO NUCLEIC-ACID STRAND ANNEALING ACTIVITY OF HETEROGENEOUS NUCLEAR RIBONUCLEOPROTEIN PROTEIN AL BY REVERSIBLE PHOSPHORYLATION

Phosphorylation in vivo of several proteins in the mammalian heterogen eous nuclear ribonucleoprotein complex (hnRNP), including A1, has been observed and proposed as a regulatory step in pre-mRNA splicing [Mayr and, S. H., Dwen, P., and Pederson, T. (1993) Proc. Natl. Acad. Sci. U .S.A. 90, 7764-7768]. We examined the ability of recombinant hnRNP pro tein Al to act as a substrate for a number of purified Ser/Thr protein kinases in vitro. A survey of seven protein kinases showed that A1 wa s heavily phosphorylated by protein kinase C (PKC) and also was phosph orylated by casein kinase II, protamine kinase, and protein kinase A. In contrast, autophosphorylation-activated protein kinase and two form s of myelin basic protein kinase failed to phosphorylate A1, Proteolys is with trypsin and V8 protease revealed that PKC phosphorylates Al at three main sites, two in the N-terminal domain (spanning residues 2-1 96) and one in the C-terminal domain (spanning residues 197-320). Amin o acid sequencing revealed that these sites were Ser(95), Ser(192), an d Ser(199); phosphorylation at Ser(192) was more abundant than at Ser( 95) and Ser(199). Phosphorylation by PKC inhibited the strand annealin g activity of A1. Protein phosphatase 2A, but not protein phosphatase 1, dephosphorylated A1 and reversed the inhibitory effect of PKC phosp horylation on the strand annealing activity. A conformational change i n the C-terminal domain of A1 was observed upon PKC phosphorylation, a nd this was associated with a decrease in A1's affinity for single-str anded polynucleotides. The results are consistent with a role of phosp horylation of A1 in regulating its strand annealing activity in vivo.