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Molecular and Cellular Biochemistry

, Volume 356, Issue 1–2, pp 191–200 | Cite as

Stimulation of CK2-dependent Grp94 phosphorylation by the nuclear localization signal peptide

  • Yoshihiko MiyataEmail author
  • Yoshihiro Yoneda
  • Ichiro Yahara
Article

Abstract

The nuclear localization signal sequence (NLS) of SV40 Large T antigen is essential and sufficient for the nuclear translocation of the protein. Phosphorylation often modulates the intracellular distribution of signaling proteins. In this study, we investigated effects of the NLS-peptide of Large T antigen on protein phosphorylation. When crude cell lysates were incubated with [γ-32P]ATP, phosphorylation of several endogenous substrates with molecular masses of 100, 80, 50, and 45 kDa by an endogenous kinase was stimulated by the addition of the wild type NLS-peptide (CPKKKRKVEDP). The mutated NLS-peptide (CPKTKRKVEDP) and the reversed NLS-peptide (PDEVKRKKKPC) are weak in the nuclear localization activity, and they only weakly stimulated phosphorylation of these substrates. The mobility of the 100 kDa phosphoprotein was indistinguishable with that of an endoplasmic reticulum (ER)-resident molecular chaperone glucose-regulated protein 94 (Grp94) belonging to the Hsp90 family, and purified Grp94 was phosphorylated by a kinase in cell lysates in an NLS-dependent fashion. The 100 kDa protein was identified as Grp94 by immunoprecipitation and reconstitution experiments. Purification of the NLS-dependent Grp94 kinase by sequential biochemical column chromatography steps resulted in isolation of two polypeptides with molecular masses of 42 and 27 kDa, which were identified as α and β subunit of protein kinase CK2, respectively, by western blotting analysis and biochemical characterization. Moreover, effect of an excess amount of GTP and V8 peptide mapping showed that the NLS-dependent Grp94 kinase in the cell lysate is identical with CK2. Surprisingly purified CK2 did phosphorylate Grp94 even without the NLS-peptide, suggesting that an additional suppressive factor is required for NLS-dependent phosphorylation of Grp94 by CK2. We suggest a possible general role for CK2-catalyzed phosphorylation in the regulation of NLS-dependent protein nuclear translocation.

Keywords

CK2 Grp94 NLS Nuclear translocation Phosphorylation Kinase 

Abbreviations

Grp94

Glucose-regulated protein 94

CK2

Protein kinase CK2

ER

Endoplasmic reticulum

NLS

Nuclear localization signal

Hsp90

Heat shock protein 90

Con A

Concanavalin A

DTT

Dithiothreitol

EDTA

Ethylenediaminetetraacetic acid

SDS-PAGE

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Notes

Acknowledgments

We thank Professor Eisuke Nishida for his support and encouragement. We would like to give our thanks to K. Kimura, H. Izumi, and T. Sakabe for their excellent technical assistance. This study was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

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Copyright information

© Springer Science+Business Media, LLC. 2011

Authors and Affiliations

  • Yoshihiko Miyata
    • 1
    • 3
    Email author
  • Yoshihiro Yoneda
    • 2
  • Ichiro Yahara
    • 3
    • 4
  1. 1.Department of Cell and Developmental Biology, Graduate School of BiostudiesKyoto UniversityKyotoJapan
  2. 2.Department of Frontier Biosciences, Graduate School of Frontier BiosciencesOsaka UniversityOsakaJapan
  3. 3.Department of Cell BiologyThe Tokyo Metropolitan Institute of Medical ScienceTokyoJapan
  4. 4.Medical & Biological Laboratories Co., LtdIna InstituteInaJapan

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