THE NEUROENDOCRINE PROTEIN 7B2 CAN BE INACTIVATED BY PHOSPHORYLATION WITHIN THE SECRETORY PATHWAY

Abstract

The prohormone convertases (PCs) play important roles in the maturation of neuropeptides and peptide hormone precursors. PC2 is the only convertase which requires the expression of another neuroendocrine protein, 7B2, for expression of enzymatic activity. In this study we determined that 7B2 can be phosphorylated in Rin cells (a rat insulinoma cell line) and cultured chromaffin cells, but not in AtT-20 cells (derived from mouse anterior pituitary). Phosphoaminoacid analysis of Rin cell 7B2 indicated the presence of phosphorylated serine and threonine. The phosphorylation of Ser115, located within the 36-residue minimally-active peptide, was confirmed by mutagenesis, although Ser115 did not represent the sole residue phosphorylated. Two independent assays were used to investigate the effect of phosphorylated 7B2 on PC2 activation: the ability of 7B2 to bind to proPC2, assessed by coimmunoprecipitation; and activation of proPC2 in a cell-free assay. Phosphorylated 7B2 was unable to bind proPC2, and phosphorylated 7B2 peptide (residues 86-121, previously shown to be the minimally active peptide for proPC2 activation) was impaired in its ability to facilitate the generation of PC2 activity in membrane fractions containing proPC2. In vitro phosphorylation experiments using Golgi membrane fractions showed that 7B2 could be phosphorylated by endogenous Golgi kinases. Golgi kinase activity was well inhibited by the broad range kinase inhibitor staurosporine and partially inhibited by the PKC inhibitor bisindolylmaleimide I, but not by the other PKA, CaM kinase II, MLCK, or PKG inhibitors tested. We conclude that phosphorylation of 7B2 functionally inactivates this protein and suggest that this may be analogous to the phosphorylating inactivation of BiP which impairs its ability to bind substrate.