title: Regulation of in vitro nucleic acid strand annealing activity of heterogeneous nuclear ribonucleoprotein protein A1 by reversible phosphorylation.
abstract: Phosphorylation in vivo of several proteins in the mammalian heterogeneous nuclear ribonucleoprotein complex (hnRNP), including A1, has been observed and proposed as a regulatory step in pre-mRNA splicing [Maryland, S. H., Dwen, P., & Pederson, T. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 7764-7768]. We examined the ability of recombinant hnRNP protein A1 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 was heavily phosphorylated by protein kinase C (PKC) and also was phosphorylated by casein kinase II, protamine kinase, and protein kinase A. In contrast, autophosphorylation-activated protein kinase and two forms of myelin basic protein kinase failed to phosphorylate A1. Proteolysis with trypsin and V8 protease revealed that PKC phosphorylates A1 at three main sites, two in the N-terminal domain (spanning residues 2-196) and one in the C-terminal domain (spanning residues 197-320). Amino acid sequencing revealed that these sites were Ser95, Ser192, and Ser199; phosphorylation at Ser192 was more abundant than at Ser95 and Ser199. Phosphorylation by PKC inhibited the strand annealing activity of A1. Protein phosphatase 2A, but not protein phosphatase 1, dephosphorylated A1 and reversed the inhibitory effect of PKC phosphorylation on the strand annealing activity. A conformational change in the C-terminal domain of A1 was observed upon PKC phosphorylation, and this was associated with a decrease in A1's affinity for single-stranded polynucleotides. The results are consistent with a role of phosphorylation of A1 in regulating its strand annealing activity in vivo.
authors.affiliation: Sealy Center for Molecular Science, University of Texas Medical Branch, Galveston 77555, USA.
authors.forename: H
authors.initials: H
authors.lastname: Idriss
authors.forename: A
authors.initials: A
authors.lastname: Kumar
authors.forename: J R
authors.initials: JR
authors.lastname: Casas-Finet
authors.forename: H
authors.initials: H
authors.lastname: Guo
authors.forename: Z
authors.initials: Z
authors.lastname: Damuni
authors.forename: S H
authors.initials: SH
authors.lastname: Wilson
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D047390:Casein Kinase II; D002942:Circular Dichroism; D016384:Consensus Sequence; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D038362:Glycogen Synthase Kinase 3; D000076245:Heterogeneous Nuclear Ribonucleoprotein A1; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D007700:Kinetics; D008322:Mammals; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010766:Phosphorylation; D006656:Protamine Kinase / Q000378:metabolism; D011487:Protein Conformation; D011493:Protein Kinase C / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism*; D054645:Protein Phosphatase 1; D054648:Protein Phosphatase 2; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D012322:RNA Precursors / Q000378:metabolism; D012326:RNA Splicing; D012332:RNA, Heterogeneous Nuclear / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012261:Ribonucleoproteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D000076245:Heterogeneous Nuclear Ribonucleoprotein A1; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D012322:RNA Precursors; D012332:RNA, Heterogeneous Nuclear; D011994:Recombinant Proteins; D012261:Ribonucleoproteins; D011494:Protein Kinases; D047390:Casein Kinase II; D006656:Protamine Kinase; D017346:Protein Serine-Threonine Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D038362:Glycogen Synthase Kinase 3; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; D054648:Protein Phosphatase 2