Phosphorylation at the Cyclin-dependent Kinases Site (Thr85) of Parathyroid Hormone-related Protein Negatively Regulates Its Nuclear Localization

doi:10.1074/jbc.274.26.18559

Phosphorylation at the Cyclin-dependent Kinases Site (Thr⁸⁵) of Parathyroid Hormone-related Protein Negatively Regulates Its Nuclear Localization*

From the St. Vincent’s Institute of Medical Research and the Department of Medicine, The University of Melbourne, 41 Victoria Parade, Fitzroy, Victoria 3065, Australia, the ^¶Cancer Research Program, The Garvan Institute of Medical Research, St. Vincent’s Hospital, 384 Victoria Street, Darlinghurst, Sydney, New South Wales 2010, Australia, and ^‖The Peter MacCallum Cancer Institute, St. Andrew’s Place, East Melbourne, Victoria 3002, Australia

Abstract

Parathyroid hormone-related protein (PTHrP) is expressed by a wide variety of cells and is considered to act as a secreted factor; however, evidence is accumulating for it to act in an intracrine manner. We have determined that PTHrP localizes to the nucleus at the G₁ phase of the cell cycle and is transported to the cytoplasm when cells divide. PTHrP contains a putative nuclear localization sequence (NLS) (residues 61–94) similar to that of SV40 T-antigen, which may be implicated in the nuclear import of the molecule. We identified that Thr⁸⁵immediately prior to the NLS of PTHrP was phosphorylated by CDC2-CDK2 and phosphorylation was cell cycle-dependent. Mutation of Thr⁸⁵ to Ala⁸⁵ resulted in nuclear accumulation of PTHrP, while mutation to Glu⁸⁵ to mimic a phosphorylated residue resulted in localization of PTHrP to the cytoplasm. Combined, the data demonstrate that the intracellular localization of PTHrP is phosphorylation- and cell cycle-dependent, and such control further supports a potential intracellular role (10, 34, 35) for PTHrP.

Footnotes

↵* This work was supported by a Program Grant (to T. J. M. and M. T. G.), Project Grants (to R. R. and B. E. K.), and a Block Grant (to B. S.) from the National Health and Medical Research Council of Australia and by the Chugai Pharmaceutical Company, Ltd., Japan (to T. J. M. and M. T. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵‡ Recipient of an Anti-Cancer Council of Victoria Postdoctoral Fellowship. Current address: Nuclear Signaling Laboratory, Dept. of Biochemistry and Molecular Biology, John Curtin School of Medical Research, Canberra, A. C. T. 2601, Australia.
↵§ National Health and Medical Research Council of Australia C. J. Martin fellow.
↵** To whom correspondence should be addressed: St. Vincent’s Institute of Medical Research, 41 Victoria Parade, Fitzroy, Victoria 3065, Australia. Tel.: 613-9288-2480; Fax: 613-9416-2676; E-mail:m.gillespie{at}medicine.unimelb.edu.au.
Abbreviations:

PTHrP

parathyroid hormone-related protein

CDK

cyclin-dependent kinase

CK2

protein kinase CK2 (formerly casein kinase II)

CLSM

confocal laser scanning microscopy

GFP

enhanced green fluorescent protein

HPLC

high pressure liquid chromatography

NLS

nuclear localization signal

NoS

nucleolar targeting signal

obrf

oligonucleotide for bone resorbing factor

DMEM

Dulbecco’s modified Eagle’s medium

FBS

fetal bovine serum

MOPS

4-morpholinepropanesulfonic acid

MALDI-TOF

matrix-assisted laser desorption ionization time-of-flight

FACS

fluorescence-activated cell sorter
- Received January 12, 1999.
- Revision received April 2, 1999.
The American Society for Biochemistry and Molecular Biology, Inc.