Post-Translational Phosphorylation of Vitellogenin

Wallace, Robin

Abstract

Vitellogenin, the yolk protein precursor, serves as an excellent model protein to study the hormonal regulation of transcriptional, translational and posttranslational events. This proposal focuses on one such posttranslational event, the phosphorylation of over 90 serine hydroxyl groups in each vitellogenin polypeptide during its translocation through the secretary apparatus of the hepatocyte. Xenopus vitellogenin microheterogeneity will first be examined by resolving vitellogenin and yolk protein polypeptides and testing whether this heterogeneity arises from differential modification of a common polypeptide or rather from slightly different primary sequences. A variety of useful substrates, both in phosphorylated and unphosphorylated form, should be concomitantly obtained during this effort. Direct evidence will be sought that vitellogenin represents the principal endogenous substrate in liver microsomes from estrogen-treated animals, and optimum procedures will be defined for the extraction and purification of vitellogenin kinase activity from such liver microsomes. During kinase purification, the existence of multiple kinases will also be explored. Purified kinase(s) will be characterized, assigned to microsomal subfractions, and related to cytosolic """"""""phosvitin kinase."""""""" Male liver microsomes will also be examined for vitellogenin kinase activity, and vitellogenin phosphorylation and processing will be examined in Xenopus oocytes microinjected with vitellogenin mRNA. These studies should accomplish several goals, including (a) elucidation of the relationship among the vitellogenin and yolk protein polypeptides and the recently described 4 vitellogenin genes in Xenopus, (b) identification of the mechanism by which vitellogenin is phosphorylated during its secretory translocation, (c) indication of the relationship, if any, between the vitellogenin kinase(s) and the ubiquitous """"""""phosvitin kinases"""""""" described by numerous laboratories and for which there is as yet no known biological function, and (d) documentation of whether vitellogenin kinase is coordinately induced in liver by estrogen or rather is a constituent enzyme of the endoplasmic reticulum/Golgi elements of all xeropus cells. In addition, these studies will allow for the first time a direct comparison between vitellogenin phosphorylation and an analogous (perhaps even homologous) system: the phosphorylation of milk protein (caseins).