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 G 1 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 Thr 85 immediately prior to the NLS of PTHrP was phosphorylated by CDC2-CDK2 and phosphorylation was cell cycle-dependent. Mutation of Thr 85 to Ala 85 resulted in nuclear accumulation of PTHrP, while mutation to Glu 85 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.Parathyroid hormone-related protein (PTHrP) 1 is widely expressed (1-3) 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 (4). Nucleolar localization of PTHrP delays apoptosis in chondrocytes (4) and increases smooth muscle cell proliferation (5). PTHrP has also been linked to the ras signaling pathway (6) and the hedgehog signaling pathways (7,8), indicating its importance in regulating growth and differentiation. PTHrP expression is cell cycle-dependent (9, 10) and PTHrP mRNA expression responds to mitogenic factors only at the G 1 phase of the cell cycle (10). Furthermore, PTHrP localizes to the nucleolus at the G 1 phase of the cell cycle (10).Cyclin-dependent kinases control the progression of the various phases of the cell cycle (11). 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 G 2 and M phases of the cell cycle. Cyclin E-CDK2 and cyclin A-CDK2 complexes are involved in the G 1 to S transition, while CDK4 and CDK6 associating with the D-type cyclins are involved in the progression through G 1 (12). In addition to direct regulation of the cell cycle, cyclins and CDKs have functions in other biological processes such as transcriptional control (13), and protein phosphorylation by CDC2 and CDK2 results in increase of affinity for the cytoplasm of some molecules containing an NLS (14).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-a...

Severe growth retardation and early lethality in mice lacking the nuclear localization sequence and C-terminus of PTH-related protein

“…The early lethality in Pth1r null mice (13) as well as in our mice lacking the nuclear localizing/C-terminal domains, may indicate that unique mechanisms may exist for PTH1R and for the nuclear localizing domain to promote optimal survival, and the absence of both regions, in the Pthrp null mice may co-operatively contribute to the more rapid death of these animals than of the mice lacking the nuclear localizing/C-terminal domains. In this respect, the latter PTHrP domains have been implicated in enhancement of proliferation (6,20,21), a function reported to require both an intact NLS and an intact C-terminal region (20), and in inhibition of apoptosis (4,6,40,41) in several cell systems in vitro. In contrast, the amino (N) terminal domain has been reported to either stimulate (42) or inhibit (6, 43) proliferation depending on the in vitro system used, and to also stimulate (44) or inhibit (45) apoptosis.…”

Constitutive c-Myb amino-terminal phosphorylation and DNA binding activity uncoupled during entry and passage through the cell cycle

“…The sequences for peptides used are: cyclin dependent kinase 2 (CDK2) peptide substrate (PTHrP 67-94 ) used to assay CDK2 activity; YLTQETNKVETYKEQPLKTPGKKKKAKP and casein kinase 2 (CK2) peptide substrate, (R3D3) used to assay CK2 enzyme activity; RRRDDDSDDD (Lam et al, 1999). Also, peptide Myb2-20 (ARRPRHSIYSSDEDDEDIE) was used to immunize rabbits.…”

Mechanism of Microtubule-facilitated “Fast Track” Nuclear Import

“…6 and Table 1). GFP-PTHrP-(82-108) showed 50% reduced maximal binding (B max of 42,900 arbitrary units) with a lower binding affinity (K d of 13.1 nM) compared with GFP-PTHrP-(66 -108), confirming that residues 66 -82 are important for interaction with human IMP␤1, although they are not sufficient to bind to IMP␤1 (41,44).…”