The subcellular localization of SF2/ASF is regulated by direct interaction with SR protein kinases (SRPKs)

Jun Koizumi, Yoshichika Okamoto, Hiroshi Onogi, Akira Maeda, Adrian R. Krainer, Masatoshi Hagiwara

Research output: Contribution to journalArticle

97 Citations (Scopus)

Abstract

Serine/arginine-rich (SR) proteins play an important role in constitutive and alternative pre-mRNA splicing. The C-terminal arginine- serine domain of these proteins, such as SF2/ASF, mediates protein-protein interactions and is phosphorylated in vivo. Using glutathione S-transferase (GST)-SF2/ASF-affinity chromatography, the SF2/ASF kinase activity was co- purified from HeLa cells with a 95-kDa protein, which was recognized by an anti-SR protein kinase (SRPK) 1 monoclonal antibody. Recombinant SRPK1 and SRPK2 bound to and phosphorylated GST-SF2/ASF in vitro. Phosphopeptide mapping showed that identical sites were phosphorylated in the pull-down kinase reaction with HeLa extracts and by recombinant SRPKs. Epitope-tagged SF2/ASF transiently expressed in COS7 cells co-immunoprecipitated with SRPKs. Deletion analysis mapped the phosphorylation sites to a region containing an (Arg-Ser)8 repeat beginning at residue 204, and far-Western analysis showed that the region is required for binding of SRPKs to SF2/ASF. Further binding studies showed that SRPKs bound unphosphorylated SF2/ASF but did not bind phosphorylated SF2/ASF. Expression of an SRPK2 kinase-inactive mutant caused accumulation of SF2/ASF in the cytoplasm. These results suggest that the formation of complexes between SF2/ASF and SRPKs, which is influenced by the phosphorylation state of SF2/ASF, may have regulatory roles in the assembly and localization of this splicing factor.

Original languageEnglish
Pages (from-to)11125-11131
Number of pages7
JournalJournal of Biological Chemistry
Volume274
Issue number16
DOIs
Publication statusPublished - 16-04-1999

Fingerprint

Protein Kinases
Serine
Arginine
Phosphorylation
Phosphotransferases
Proteins
Glutathione Transferase
Affinity chromatography
Recombinant proteins
Phosphopeptides
RNA Precursors
Affinity Chromatography
HeLa Cells
Recombinant Proteins
Epitopes
Carrier Proteins
Cytoplasm
Monoclonal Antibodies

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Koizumi, Jun ; Okamoto, Yoshichika ; Onogi, Hiroshi ; Maeda, Akira ; Krainer, Adrian R. ; Hagiwara, Masatoshi. / The subcellular localization of SF2/ASF is regulated by direct interaction with SR protein kinases (SRPKs). In: Journal of Biological Chemistry. 1999 ; Vol. 274, No. 16. pp. 11125-11131.
@article{a80c2838040a4fabb2139891ff4414c1,
title = "The subcellular localization of SF2/ASF is regulated by direct interaction with SR protein kinases (SRPKs)",
abstract = "Serine/arginine-rich (SR) proteins play an important role in constitutive and alternative pre-mRNA splicing. The C-terminal arginine- serine domain of these proteins, such as SF2/ASF, mediates protein-protein interactions and is phosphorylated in vivo. Using glutathione S-transferase (GST)-SF2/ASF-affinity chromatography, the SF2/ASF kinase activity was co- purified from HeLa cells with a 95-kDa protein, which was recognized by an anti-SR protein kinase (SRPK) 1 monoclonal antibody. Recombinant SRPK1 and SRPK2 bound to and phosphorylated GST-SF2/ASF in vitro. Phosphopeptide mapping showed that identical sites were phosphorylated in the pull-down kinase reaction with HeLa extracts and by recombinant SRPKs. Epitope-tagged SF2/ASF transiently expressed in COS7 cells co-immunoprecipitated with SRPKs. Deletion analysis mapped the phosphorylation sites to a region containing an (Arg-Ser)8 repeat beginning at residue 204, and far-Western analysis showed that the region is required for binding of SRPKs to SF2/ASF. Further binding studies showed that SRPKs bound unphosphorylated SF2/ASF but did not bind phosphorylated SF2/ASF. Expression of an SRPK2 kinase-inactive mutant caused accumulation of SF2/ASF in the cytoplasm. These results suggest that the formation of complexes between SF2/ASF and SRPKs, which is influenced by the phosphorylation state of SF2/ASF, may have regulatory roles in the assembly and localization of this splicing factor.",
author = "Jun Koizumi and Yoshichika Okamoto and Hiroshi Onogi and Akira Maeda and Krainer, {Adrian R.} and Masatoshi Hagiwara",
year = "1999",
month = "4",
day = "16",
doi = "10.1074/jbc.274.16.11125",
language = "English",
volume = "274",
pages = "11125--11131",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "16",

}

The subcellular localization of SF2/ASF is regulated by direct interaction with SR protein kinases (SRPKs). / Koizumi, Jun; Okamoto, Yoshichika; Onogi, Hiroshi; Maeda, Akira; Krainer, Adrian R.; Hagiwara, Masatoshi.

In: Journal of Biological Chemistry, Vol. 274, No. 16, 16.04.1999, p. 11125-11131.

Research output: Contribution to journalArticle

TY - JOUR

T1 - The subcellular localization of SF2/ASF is regulated by direct interaction with SR protein kinases (SRPKs)

AU - Koizumi, Jun

AU - Okamoto, Yoshichika

AU - Onogi, Hiroshi

AU - Maeda, Akira

AU - Krainer, Adrian R.

AU - Hagiwara, Masatoshi

PY - 1999/4/16

Y1 - 1999/4/16

N2 - Serine/arginine-rich (SR) proteins play an important role in constitutive and alternative pre-mRNA splicing. The C-terminal arginine- serine domain of these proteins, such as SF2/ASF, mediates protein-protein interactions and is phosphorylated in vivo. Using glutathione S-transferase (GST)-SF2/ASF-affinity chromatography, the SF2/ASF kinase activity was co- purified from HeLa cells with a 95-kDa protein, which was recognized by an anti-SR protein kinase (SRPK) 1 monoclonal antibody. Recombinant SRPK1 and SRPK2 bound to and phosphorylated GST-SF2/ASF in vitro. Phosphopeptide mapping showed that identical sites were phosphorylated in the pull-down kinase reaction with HeLa extracts and by recombinant SRPKs. Epitope-tagged SF2/ASF transiently expressed in COS7 cells co-immunoprecipitated with SRPKs. Deletion analysis mapped the phosphorylation sites to a region containing an (Arg-Ser)8 repeat beginning at residue 204, and far-Western analysis showed that the region is required for binding of SRPKs to SF2/ASF. Further binding studies showed that SRPKs bound unphosphorylated SF2/ASF but did not bind phosphorylated SF2/ASF. Expression of an SRPK2 kinase-inactive mutant caused accumulation of SF2/ASF in the cytoplasm. These results suggest that the formation of complexes between SF2/ASF and SRPKs, which is influenced by the phosphorylation state of SF2/ASF, may have regulatory roles in the assembly and localization of this splicing factor.

AB - Serine/arginine-rich (SR) proteins play an important role in constitutive and alternative pre-mRNA splicing. The C-terminal arginine- serine domain of these proteins, such as SF2/ASF, mediates protein-protein interactions and is phosphorylated in vivo. Using glutathione S-transferase (GST)-SF2/ASF-affinity chromatography, the SF2/ASF kinase activity was co- purified from HeLa cells with a 95-kDa protein, which was recognized by an anti-SR protein kinase (SRPK) 1 monoclonal antibody. Recombinant SRPK1 and SRPK2 bound to and phosphorylated GST-SF2/ASF in vitro. Phosphopeptide mapping showed that identical sites were phosphorylated in the pull-down kinase reaction with HeLa extracts and by recombinant SRPKs. Epitope-tagged SF2/ASF transiently expressed in COS7 cells co-immunoprecipitated with SRPKs. Deletion analysis mapped the phosphorylation sites to a region containing an (Arg-Ser)8 repeat beginning at residue 204, and far-Western analysis showed that the region is required for binding of SRPKs to SF2/ASF. Further binding studies showed that SRPKs bound unphosphorylated SF2/ASF but did not bind phosphorylated SF2/ASF. Expression of an SRPK2 kinase-inactive mutant caused accumulation of SF2/ASF in the cytoplasm. These results suggest that the formation of complexes between SF2/ASF and SRPKs, which is influenced by the phosphorylation state of SF2/ASF, may have regulatory roles in the assembly and localization of this splicing factor.

UR - http://www.scopus.com/inward/record.url?scp=0033574564&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0033574564&partnerID=8YFLogxK

U2 - 10.1074/jbc.274.16.11125

DO - 10.1074/jbc.274.16.11125

M3 - Article

VL - 274

SP - 11125

EP - 11131

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 16

ER -