Identification, localization, and primary structure of CAP-23, a particle-bound cytosolic protein of early development

Abstract

We report the identification of CAP-23, a novel particle-bound cytosolic protein associated with developing cells in both mammalian and avian tissues. CAP-23 was a substrate for purified protein kinase C (PKC) in vitro, and the protein was phosphorylated in a PMA-sensitive manner in cultured cells, indicating that it is a PKC substrate in situ. cDNA coding for chick CAP-23 was isolated. The deduced sequence revealed an unusual amino acid composition that strikingly resembled that of rat GAP-43, a growth-associated neuron-specific PKC substrate. Further predicted features of CAP-23 included a PKC phosphorylation site at Ser-6, and the presence of basic NH2- and COOH-terminal domains. CAP-23 was encoded by an mRNA of approximately 1.5 kb, whose distribution during chick development resembled that of the corresponding protein. Southern blot analysis revealed the presence of a single main hybridizing species in the chick genome. The distribution of CAP-23 during development was analyzed with Western blots and by immunofluorescence on tissue sections. In cultured cells the protein appeared to be distributed in a regular spotted pattern below the entire cell surface. In early chick embryos (E2), CAP-23 was present in most if not all cells. The protein then became progressively restricted to only some developing tissues and to only certain cells in these tissues. In most tissues CAP-23 levels fell below detection limits between E15 and E19. Highest levels of the protein were found in the nervous system, where CAP-23 levels peaked around E18, and where elevated levels were still detectable at birth.