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

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 (10Lam M.H.C. Olsen S.L. Rankin W.A. Ho P.W.M. Martin T.J. Gillespie M.T. Moseley J.M. J. Cell. Physiol. 1997; 173: 433-446Crossref PubMed Scopus (65) Google Scholar, 34Amizuka N. Warshawsky H. Henderson J.E. Goltzman D. Karaplis A.C. J. Cell Biol. 1994; 126: 1611-1623Crossref PubMed Scopus (423) Google Scholar, 35Amizuka N. Henderson J.E. Hoshi K. Warshawsky H. Ozawa H. Goltzman D. Karaplis A.C. Endocrinology. 1996; 137: 5055-5067Crossref PubMed Scopus (121) Google Scholar) for PTHrP. 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 (10Lam M.H.C. Olsen S.L. Rankin W.A. Ho P.W.M. Martin T.J. Gillespie M.T. Moseley J.M. J. Cell. Physiol. 1997; 173: 433-446Crossref PubMed Scopus (65) Google Scholar, 34Amizuka N. Warshawsky H. Henderson J.E. Goltzman D. Karaplis A.C. J. Cell Biol. 1994; 126: 1611-1623Crossref PubMed Scopus (423) Google Scholar, 35Amizuka N. Henderson J.E. Hoshi K. Warshawsky H. Ozawa H. Goltzman D. Karaplis A.C. Endocrinology. 1996; 137: 5055-5067Crossref PubMed Scopus (121) Google Scholar) for PTHrP. Parathyroid hormone-related protein (PTHrP) 1The abbreviations used are: 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. is widely expressed (1Goltzman D. Henderson J.E. Kaiser S. Kremer R. Rabbani S.A. Hendy G.N. J. Clin. Invest. 1992; 15: 43-49Google Scholar, 2Moseley J.M. Gillespie M.T. Crit. Rev. Clin. Lab. Sci. 1995; 32: 299-343Crossref PubMed Scopus (94) Google Scholar, 3Philbrick W.M. Wysolmerski J.J. Galbraith S. Holt E. Orloff J.J. Yang K.H. Vasavada R.C. Weir E.C. Broadus A.E. Stewart A.F. Physiol. Rev. 1996; 76: 127-173Crossref PubMed Scopus (523) Google Scholar) and acts as a paracrine, and possibly an autocrine and intracrine, factor. However, its intracellular roles have not been fully defined. Recently a nucleolar localization signal (NLS) was identified within PTHrP and deletion of this motif prevented PTHrP from entering the nucleolus, maintaining it as a cytoplasmic protein (4Henderson J.E. Amizuka N. Warshawsky H. Biasotto D. Lanske B.M. Karaplis A.C. Mol. Cell. Biol. 1995; 15: 4064-4075Crossref PubMed Scopus (307) Google Scholar). Nucleolar localization of PTHrP delays apoptosis in chondrocytes (4Henderson J.E. Amizuka N. Warshawsky H. Biasotto D. Lanske B.M. Karaplis A.C. Mol. Cell. Biol. 1995; 15: 4064-4075Crossref PubMed Scopus (307) Google Scholar) and increases smooth muscle cell proliferation (5Massfelder T. Dann P. Wu T.L. Vasavada R. Helwig J.-J. Stewart A.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13630-13635Crossref PubMed Scopus (208) Google Scholar). PTHrP has also been linked to the ras signaling pathway (6Li X. Drucker D.J. J. Biol. Chem. 1994; 269: 6263-6266Abstract Full Text PDF PubMed Google Scholar) and the hedgehog signaling pathways (7Lanske B.M. Karaplis A.C. Lee K. Luz A. Vortkamp A. Pirro A. Karperien M. Defize L.H.K. Ho C. Mulligan R.M. Abou-Samra A.B. Juppner H. Segre G.V. Kronenberg H.M. Science. 1996; 273: 663-666Crossref PubMed Scopus (1135) Google Scholar, 8Vortkamp A. Lee K. Lanske B.M. Segre G.V. Kronenberg H.M. Tabin C. Science. 1996; 273: 613-622Crossref PubMed Scopus (1659) Google Scholar), indicating its importance in regulating growth and differentiation. PTHrP expression is cell cycle-dependent (9Okano K. Pirola C.J. Wang H.M. Forrester J.S. Fagin J.A. Clemens T.L. Endocrinology. 1995; 136: 1782-1789Crossref PubMed Google Scholar, 10Lam M.H.C. Olsen S.L. Rankin W.A. Ho P.W.M. Martin T.J. Gillespie M.T. Moseley J.M. J. Cell. Physiol. 1997; 173: 433-446Crossref PubMed Scopus (65) Google Scholar) and PTHrP mRNA expression responds to mitogenic factors only at the G1 phase of the cell cycle (10Lam M.H.C. Olsen S.L. Rankin W.A. Ho P.W.M. Martin T.J. Gillespie M.T. Moseley J.M. J. Cell. Physiol. 1997; 173: 433-446Crossref PubMed Scopus (65) Google Scholar). Furthermore, PTHrP localizes to the nucleolus at the G1 phase of the cell cycle (10Lam M.H.C. Olsen S.L. Rankin W.A. Ho P.W.M. Martin T.J. Gillespie M.T. Moseley J.M. J. Cell. Physiol. 1997; 173: 433-446Crossref PubMed Scopus (65) Google Scholar). Cyclin-dependent kinases control the progression of the various phases of the cell cycle (11Norbury C. Nurse P. Annu. Rev. Biochem. 1992; 61: 441-470Crossref PubMed Google Scholar). CDKs are activated at different phases of the cell cycle by the formation of cyclin-CDK complex and deactivated when their cyclin partner is degraded. The prototype CDK is CDC2, which associates with cyclin B and regulates the transition between the G2 and M phases of the cell cycle. Cyclin E-CDK2 and cyclin A-CDK2 complexes are involved in the G1to S transition, while CDK4 and CDK6 associating with the D-type cyclins are involved in the progression through G1 (12Pines J. Biochem. J. 1995; 308: 697-711Crossref PubMed Scopus (498) Google Scholar). In addition to direct regulation of the cell cycle, cyclins and CDKs have functions in other biological processes such as transcriptional control (13Poon R.Y.C. Hunter T. Curr. Biol. 1995; 5: 1243-1247Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar), and protein phosphorylation by CDC2 and CDK2 results in increase of affinity for the cytoplasm of some molecules containing an NLS (14Jans D.A. Biochem. J. 1995; 311: 705-716Crossref PubMed Scopus (179) Google Scholar). A stretch of basic residues, or a pair of basic residues separated by a 10–12 amino acid spacer to form a bipartite NLS, characterizes NLSs. The archetypal protein used in nuclear localization studies is the SV40 T-antigen where nuclear/cytoplasmic localization is regulated by phosphorylation. CK2 and/or CDC2-CDK2 phosphorylations at sites near the NLS determine the rate and amount of localization within the nucleus in an NLS-dependent manner (15Rihs H.-P. Jans D.A. Fan H. Peters R. EMBO J. 1991; 10: 633-639Crossref PubMed Scopus (302) Google Scholar, 16Jans D.A. Ackermann M.J. Bischoff J.R. Beach D.H. Peters R. J. Cell Biol. 1991; 115: 1203-1212Crossref PubMed Scopus (183) Google Scholar, 17Jans D.A. Jans P. Oncogene. 1994; 9: 2961-2968PubMed Google Scholar). The combination of a CK2 site, a CDC2-CDK2 site and an NLS constitute a CcN motif, which is involved in phosphorylation-regulated nuclear localization. The CcN motif is conserved in a number of nuclear localized proteins, including mammalian c-MYC, p53, and mouse c-ABL IV and A-MYB (14Jans D.A. Biochem. J. 1995; 311: 705-716Crossref PubMed Scopus (179) Google Scholar, 18Jans D.A. Hübner S. Physiol. Rev. 1996; 76: 652-658Crossref Scopus (389) Google Scholar). PTHrP contains a putative CcN motif similar to SV40 T-antigen, consisting of a CK2 site, and a CDC2-CDK2 site immediately before the NLS (Fig. 2). Additionally, a putative nucleolar targeting sequence (NoS) is located immediately C-terminal to the NLS. In the present work using overexpressing cells, PTHrP was found to be phosphorylated in vivo. Using in vitro assays cyclin E-CDK2, cyclin A-CDK2 and cyclin B-CDC2 phosphorylated PTHrP at Thr85, which is immediately prior to its NLS. With the use of wild type and mutant green fluorescent protein GFP-PTHrP fusion proteins overexpressed in HaCaT cells, it was found that phosphorylation of Thr85 resulted in cytoplasmic retention/nuclear exclusion. The results of these studies indicate a cell cycle-dependent nuclear exclusion of PTHrP from the start of S phase to mitosis, providing support for a tightly regulated nuclear function for PTHrP at G1. The spontaneously immortalized human keratinocyte cell line, HaCaT, which expresses PTHrP, was a kind gift from Professor N. E. Fusenig and was grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS at 37 °C and equilibrated with 5% CO2, as described previously (19Heath J.K. Southby J. Fukumoto S. O'Keeffe L.M. Martin T.J. Gillespie M.T. Biochem. J. 1995; 307: 159-167Crossref PubMed Scopus (53) Google Scholar). The human T lymphoid cell line (CEM) was grown in RPMI 1640 medium supplemented with 10% fetal bovine serum, 50 units/ml of penicillin, and 50 μg/ml streptomycin. 70% confluent HaCaT cells fixed and stained with a fluorescein-labeled monoclonal antibody against amino terminus PTHrP as described previously (10Lam M.H.C. Olsen S.L. Rankin W.A. Ho P.W.M. Martin T.J. Gillespie M.T. Moseley J.M. J. Cell. Physiol. 1997; 173: 433-446Crossref PubMed Scopus (65) Google Scholar, 20Rankin W. Murphy B. Grill V. Ho P.W.M. Moseley J.M. Martin T.J. Bone (N. Y.). 1995; 16: 205SGoogle Scholar). The cells were counter-stained with the nuclei acid-specific dye, propidium iodide. Images were collected and generated on a confocal laser scanning microscope (CLSM; Bio-Rad MRC 1024, Bio-Rad Microscopy Division, Hemel Hempstead Herts, United Kingdom). The histidine-tagged protein A-bovine cyclin A complimentary DNA (cDNA) in a pET16b vector and the human p34CDK2 cDNA in pGEX was a generous gift from Dr. T. Hunt (ICRF Clare Hall Laboratories, UK). GST-p34CDK2 and histidine-tagged protein A-cyclin A were overexpressed inEscherichia coli strain BL21(DE3) and activated as described in R.Y.C. K. P. Hunt T. J. EMBO J. PubMed Scopus Google Scholar). for cyclin A from Dr. The and cyclin gift from Dr. The were by and the vector and A were with an and the CDK2 was with an The of were by were generated to the and cells were with high cyclin and CDK for the cells were and cyclin-CDK complexes using affinity for human B and CDC2 were a kind gift from Dr. and were expressed and as described kinase assays were in a containing 50 MOPS, and of and μg/ml to The was in a containing 50 MOPS, and The were for at of the and and as described in Scholar). of PTHrP phosphorylation by various cyclin-CDK and studies of of PTHrP and of were were by 50 and of were at the cyclin-CDK of and were with of cyclin E-CDK2 and cyclin were for cyclin the was for studies cyclin E-CDK2 and cyclin A-CDK2 were for and cyclin was for were by of the and with acid and in was used to determine is a for cyclin E-CDK2, cyclin and cyclin of was used as in vitro for cyclin E-CDK2, cyclin and cyclin and of was used as a while of was used as control and also to demonstrate the of PTHrP as a for these The of cyclin cyclin and cyclin not not in a studies of PTHrP assays for cyclin B-CDC2 were was from the for further studies were as described The used was and assays were using cyclin E-CDK2 cyclin A-CDK2 and cyclin B-CDC2 assays for cyclin B-CDC2 were was from the for further in a of was used as in vitro for cyclin E-CDK2, cyclin and cyclin and of was used as a while of was used as control and also to demonstrate the of PTHrP as a for these The of cyclin cyclin and cyclin not not studies were as described The used was and assays were using cyclin E-CDK2 cyclin A-CDK2 and cyclin B-CDC2 2). and and CK2 were using the and and as described in Scholar). and were expressed in E. as described in P. H. Grill V. J. Moseley J.M. Martin T.J. J. Biol. Chem. Full Text PDF PubMed Google Scholar). and protein were determined by amino acid acid and proteins were phosphorylated as described in the protein kinase for and were used for and The were in a of for at and by of of from phosphorylated and proteins was by to a Laboratories, that was equilibrated with of was using of while was with acid in were and to a activated previously to and to the were with of with of was phosphorylated as described using and on a phase using a with a of in acid at a rate of and a rate of at and an amount of was also to the and to to phosphorylated and PTHrP were collected and their by matrix-assisted laser desorption ionization on a using acid as the of proteins or phosphorylated with were at 37 °C for with of in of and 10% The were using through a phase and with as described of were used to with the of phosphorylated PTHrP was as described by and Hunter K. Hunter T. J. PubMed Google Scholar). The were in a or an for of with of DNA for was in to a of 50 and to cells that were grown to in a and in of 10% The cells were in at with the between and using a Laboratories, The cells were of 10% FBS and to for The medium was the cells The cells were to for before were cells were in DMEM, containing 10% FBS for of in the form of acid in was to the medium and for The cells were in and in of 50 were in of the and with for at The were and of antibody was to the and at complexes were collected with of protein with and by were The was and was using that in to of were from the with and by of cells was in the with a at The was in RPMI medium with 5% fetal bovine serum, and The G1 cell was in RPMI medium with 10% fetal bovine serum, and were collected at Cell cycle of of cells were determined by with propidium and using the in a was used to phosphorylated and of PTHrP. the NLS with the The and sites at the to was to amino and for Thr85, and Thr85 to and when used in with generated a site at the The fusion protein control was generated using and were generated using of the with the cDNA for PTHrP Moseley J.M. H. Martin T.J. Science. PubMed Scopus Google Scholar) as and were in C-terminal to the in the vector using and The of DNA was by DNA using a DNA DNA was and HaCaT cells using the as described in J. T. A Scholar). were counter-stained with the dye, propidium or with the dye, Images were by and of PTHrP expression in a of HaCaT cells that PTHrP was localized to the nucleolus and the cytoplasm in cells that a G1 phase (Fig. and In cells that a G2 and M which have a nuclear PTHrP was not in the nucleus was expressed at in the cytoplasm (Fig. A and and PTHrP expression was in cells (Fig. A and of this at the sequence of the PTHrP protein with that of various nuclear The amino acid sequence of PTHrP with the CcN of SV40 T-antigen, p53, c-MYC, and A-MYB (13Poon R.Y.C. Hunter T. Curr. Biol. 1995; 5: 1243-1247Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 17Jans D.A. Jans P. Oncogene. 1994; 9: 2961-2968PubMed Google Scholar). The CcN motif a putative CDK site at C-terminal to the CcN is a putative with of basic amino an which is found in proteins such as Lee J. Biol. Chem. Full Text PDF PubMed Google Scholar), W.A. A. J. PubMed Google Scholar), and H. H. T. M. M. Cell. Full Text PDF PubMed Scopus Google Scholar). the combination of of the NLS and the constitute a bipartite NLS (Fig. where of basic residues are separated by a acid spacer J. C. Cell. 1991; Full Text PDF PubMed Scopus Google Scholar, M. C.J. J. Biol. Chem. 1991; Full Text PDF PubMed Google Scholar). of these it was to determine PTHrP be phosphorylated by PTHrP and PTHrP proteins were with cyclin and proteins were to a of and phosphorylated using μg/ml cyclin and the were phosphorylated by cyclin while the PTHrP and were not phosphorylated (Fig. Cyclin A-CDK2 phosphorylated only PTHrP proteins and containing the putative phosphorylation motif results indicate that the phosphorylation site of PTHrP within amino and Thr85, the only phosphorylated amino acid A-CDK2 PTHrP between residues and and or and were phosphorylated with μg/ml cyclin A-CDK2 as described on the is in of CK2 was used as a are the of phosphorylated with cyclin A-CDK2 was on a phase and the by The of the were in with the of phosphorylated which is and which is Thr85 was to be the CDC2-CDK2 phosphorylation site of PTHrP by of of cyclin that and the CDC2-CDK2 phosphorylation In to determine cyclin-CDK phosphorylated PTHrP, a of expressed and activated were used to in vitro Cyclin E-CDK2, cyclin and cyclin The cyclin-CDK cyclin cyclin and cyclin not not that PTHrP be phosphorylated in of the cell cycle studies the of as a to cyclin E-CDK2, cyclin and cyclin B-CDC2 were as described The for cyclin E-CDK2 was for cyclin A-CDK2 and for cyclin B-CDC2 and was in the for for cyclin E-CDK2 was cyclin A-CDK2 and cyclin B-CDC2 of activated and M cyclin E-CDK2, cyclin and cyclin phosphorylated was used as a control for kinase and also to demonstrate the of PTHrP as a with a CDC2-CDK2 was used as a control and was not phosphorylated by of the cyclin-CDK as not be between of the cyclin-CDK assays as the were of different and also different determine phosphorylation of was cell cycle cell of cells were collected at various of the cell cycle G1 by and used to with the kinase in vitro this in vitro for kinase using as that kinase was at G1 as the cells on to S (Fig. and to at G2 and M (Fig. In the of CK2 with as a was the cell cycle (Fig. that PTHrP was phosphorylated in cells with were from the cell with an antibody against resulted in a protein at to this was and by the (Fig. to that of cyclin Thr85 to be the site of phosphorylation. The in was to of this resulted in a of the in the at the CDC2-CDK2 site to the SV40 T-antigen NLS the rate and amount of nuclear localization of this protein D.A. Ackermann M.J. Bischoff J.R. Beach D.H. Peters R. J. Cell Biol. 1991; 115: 1203-1212Crossref PubMed Scopus (183) Google Scholar). In to determine this is the also with PTHrP, GFP-PTHrP fusion containing wild type and of Thr85 were Thr85 was to Ala85 to phosphorylation at this site or to Glu85 to mimic the of a phosphorylated fusion proteins the NLS and the putative (Fig. The vector fusion was used as a vector control (Fig. at the terminus to was used as a control (Fig. HaCaT cells with the vector (Fig. to at the terminus nuclear and nucleolar localization (Fig. as as cytoplasmic localization in some cells (Fig. indicating regulation of its localization the cell cycle. A similar of localization was in cells, indicating that not not HaCaT cells with the wild type fusion protein cytoplasmic and nuclear with localization to the nucleus (Fig. with nuclear localization with some cytoplasmic localization while cells that were with cytoplasmic localization only (Fig. A of cells from were with the cells for GFP-PTHrP localization to the nucleus or cytoplasm (Fig. the cells, of cells nuclear and of cells also cytoplasmic localization. of cells with the mutant nuclear while of the cells cytoplasmic localization. In cells with the cytoplasmic localization and only of these nuclear data indicate that when phosphorylation of it results in nuclear accumulation of PTHrP, while in an PTHrP is nuclear is the of an that a phosphorylation-regulated NLS and localization that it acts proteins to such have intracellular functions such as proteins SV40 and (14Jans D.A. Biochem. J. 1995; 311: 705-716Crossref PubMed Scopus (179) Google D.A. J. 1994; PubMed Scopus Google Scholar). PTHrP identified a putative CcN motif similar to of SV40 T-antigen, c-MYC, p53, and these PTHrP contains a CDC2-CDK2 phosphorylation site near the putative NLS. We that PTHrP was phosphorylated by CDC2-CDK2 at Thr85 in the CcN motif and that this phosphorylation in cells overexpressing PTHrP. of residues in the of the NLS in other proteins, such as SV40 T-antigen, their (14Jans D.A. Biochem. J. 1995; 311: 705-716Crossref PubMed Scopus (179) Google Scholar, 16Jans D.A. Ackermann M.J. Bischoff J.R. Beach D.H. Peters R. J. Cell Biol. 1991; 115: 1203-1212Crossref PubMed Scopus (183) Google Scholar). at the CDK site to the NLS these proteins from a and to the cytoplasm (14Jans D.A. Biochem. J. 1995; 311: 705-716Crossref PubMed Scopus (179) Google Scholar, 16Jans D.A. Ackermann M.J. Bischoff J.R. Beach D.H. Peters R. J. Cell Biol. 1991; 115: 1203-1212Crossref PubMed Scopus (183) Google Scholar). phosphorylation of PTHrP results in a of PTHrP, GFP-PTHrP expression in which Thr85 was to Ala85 or residues were to phosphorylation or to mimic a phosphorylated studies indicate that PTHrP in a is in the while in the phosphorylated PTHrP is from the nucleus and is In with proteins a CDK phosphorylation site immediately to a NLS (14Jans D.A. Biochem. J. 1995; 311: 705-716Crossref PubMed Scopus (179) Google Scholar, 16Jans D.A. Ackermann M.J. Bischoff J.R. Beach D.H. Peters R. J. Cell Biol. 1991; 115: 1203-1212Crossref PubMed Scopus (183) Google Scholar), phosphorylation at this site NLS function (14Jans D.A. Biochem. J. 1995; 311: 705-716Crossref PubMed Scopus (179) Google Scholar), and this also to be for PTHrP. CDC2-CDK2 phosphorylation was found to at S phase and at the phase of the cell cycle, with the localization of cyclin A and to the nucleus J. Hunter T. J. Cell Biol. 1991; 115: PubMed Scopus Google Scholar), that PTHrP be phosphorylated and from the nucleus at these In that PTHrP was localized to the nucleus further the that cell nuclear localization of PTHrP may be regulated by CDK phosphorylation. evidence has that PTHrP acts as a in with and possibly also in it acts as a in J.M. Gillespie M.T. Crit. Rev. Clin. Lab. Sci. 1995; 32: 299-343Crossref PubMed Scopus (94) Google Scholar, 3Philbrick W.M. Wysolmerski J.J. Galbraith S. Holt E. Orloff J.J. Yang K.H. Vasavada R.C. Weir E.C. Broadus A.E. Stewart A.F. Physiol. Rev. 1996; 76: 127-173Crossref PubMed Scopus (523) Google Scholar). The that PTHrP is localized to the nucleus and nucleolus that PTHrP may have intracellular roles (10Lam M.H.C. Olsen S.L. Rankin W.A. Ho P.W.M. Martin T.J. Gillespie M.T. Moseley J.M. J. Cell. Physiol. 1997; 173: 433-446Crossref PubMed Scopus (65) Google Scholar, 34Amizuka N. Warshawsky H. Henderson J.E. Goltzman D. Karaplis A.C. J. Cell Biol. 1994; 126: 1611-1623Crossref PubMed Scopus (423) Google Scholar, 35Amizuka N. Henderson J.E. Hoshi K. Warshawsky H. Ozawa H. Goltzman D. Karaplis A.C. Endocrinology. 1996; 137: 5055-5067Crossref PubMed Scopus (121) Google Scholar). A for PTHrP may as a of as a of PTHrP within a cell PTHrP or in a cell as a of to its and of the complex with nuclear localization. The control for intracellular of PTHrP, and nuclear localization is not PTHrP phosphorylation and cell cycle-dependent nuclear it is that PTHrP may have a nuclear possibly to growth and differentiation. for this results from the that intracellular PTHrP increases proliferation of smooth cells (5Massfelder T. Dann P. Wu T.L. Vasavada R. Helwig J.-J. Stewart A.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13630-13635Crossref PubMed Scopus (208) Google Scholar). is that other secreted growth such as and basic growth growth and D.A. PubMed Scopus Google Scholar, T. J. Cell Biol. PubMed Scopus Google Scholar), which basic amino acid to of a NLS, may also regulated intracellular localization similar to that of PTHrP. We Rankin for the monoclonal antibody against PTHrP, for with and for and amino acid and Dr. Jans for this