PREPROPARATHYROID HORMONE-RELATED PROTEIN, A SECRETED PEPTIDE, IS A SUBSTRATE FOR THE UBIQUITIN PROTEOLYTIC SYSTEM

Parathyroid hormone-related protein (PTHrP) is an important causal fac tor of hypercalcemia associated with malignancy. PTHrP also modulates cell growth and differentiation of normal cells through mechanisms tha t include binding to cell surface-specific receptors as well as by pos sible intracellular routes. To understand the regulation of intracellu lar PTHrP expression, posttranslational processing of PTHrP was invest igated. Using cell-free translations it was shown that PTHrP can be li gated efficiently to multiple ubiquitin moieties. Both conjugation to ubiquitin and degradation of pre pro-PTHrP synthesized in vitro were A TP dependent. Translation in vitro in the presence of the proteasome i nhibitor MG-132 abolished the degradation of PTHrP. Treatment of cells , cotransfected with hemagglutinin-tagged ubiquitin and histidine-tagg ed prepro-PTHrP, with MG 132, led to the accumulation of ubiquitinated prepro-PTHrP, Deletion mutagenesis experiments indicated that both th e prepro secretory domain and a PEST (amino acid residues Pro (P), Glu (E), and/or Asp (D), Ser (S), and Thr (T)) motif in the COOH-terminal region of the protein were not required as cis-acting determinants fo r ubiquitination. This is the first report of a wild-type secretory po lypeptide serving as a substrate of the ubiquitin proteolytic pathway. These results suggest that the ubiquitin dependent proteolytic pathwa y is involved in regulating the metabolic stability of intracellular P THrP, and this regulation may be an important mechanism for modulating its effects on cell growth and differentiation.