Phosphorylation of human hnRNP protein A1 abrogates in vitro strand annealing activity.
In HeLa cells metabolically labeled in vivo with [32P] orthophosphate in the presence of okadaic acid the concentration of phosphorylated A1 protein was increased significantly as compared to controls. Purified recombinant hnRNP protein A1 served as an excellent substrate in vitro for the catalytic subunit of cAMP-dependent protein kinase (PKA) and for casein kinase II (CKII). Thin layer electrophoresis of A1 acid hydrolysates showed the protein to be phosphorylated exclusively on serine residue by both kinases. V8 phosphopeptide maps revealed that the target site(s) of in vitro phosphorylation are located in the C-terminal region of A1. Phosphoamino acid sequence analysis and site directed mutagenesis identified Ser 199 as the sole phosphoamino acid in the protein phosphorylated by PKA. Phosphorylation introduced by PKA resulted in the suppression of the ability of protein A1 to promote strand annealing in vitro, without any detectable effect on its nucleic acid binding capacity. This finding indicates that phosphorylation of a single serine residue in the C-terminal domain may significantly alter the properties of protein A1.
Cobianchi
F
F
Istituto di Genetica Biochimica ed Evoluzionistica, CNR, Pavia, Italy.
Calvio
C
C
Stoppini
M
M
Buvoli
M
M
Riva
S
S
D000595:Amino Acid Sequence; D001483:Base Sequence; D047388:Casein Kinases; D004268:DNA-Binding Proteins / Q000502:physiology; D006367:HeLa Cells; D000076245:Heterogeneous Nuclear Ribonucleoprotein A1; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D009693:Nucleic Acid Hybridization; D010750:Phosphoproteins / Q000502:physiology; D010766:Phosphorylation; D011494:Protein Kinases; D011994:Recombinant Proteins; D012261:Ribonucleoproteins / Q000737:chemistry* / Q000502:physiology*; D012694:Serine
D004268:DNA-Binding Proteins; D000076245:Heterogeneous Nuclear Ribonucleoprotein A1; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D010750:Phosphoproteins; D011994:Recombinant Proteins; D012261:Ribonucleoproteins; D012694:Serine; D011494:Protein Kinases; D047388:Casein Kinases