35H, A SEQUENCE ISOLATED AS A PROTEIN-KINASE-C BINDING-PROTEIN, IS A NOVEL MEMBER OF THE ADDUCIN FAMILY

We recently cloned a partial cDNA (35H) for a protein kinase C (PKC) b inding protein from a rat kidney cDNA library and demonstrated that it is a PKC substrate in vitro (Chapline, C., Ramsay, K., Klauck, T., an d Jaken, S. (1993) J. Biol. Chem. 268, 6858-6861). Additional library screening and 5' rapid amplification of cDNA ends were used to obtain the complete open reading frame. Amino acid sequence analysis, DNA seq uence analysis, and Northern analysis indicate that 35H is a unique cD NA related to alpha- and beta-adducins. Antisera prepared to the 35H b acterial fusion protein recognized two polypeptides of 80 and 90 kDa o n immunoblots of kidney homogenates and cultured renal proximal tubule epithelial cell extracts. The 35H-related proteins were similar to al pha- and beta-adducins in that they were preferentially recovered in t he Triton X-100-insoluble (cytoskeletal, CSK) fraction of cell extract s and were predominantly localized to cell borders. Phorbol esters sti mulated phosphorylation of CSK 35H proteins, thus emphasizing that seq uences isolated according to PKC binding activity in vitro are also PK C substrates in vivo. The phosphorylated forms of the 35H proteins wer e preferentially recovered in the soluble fraction, thus demonstrating that phosphorylation regulates their CSK association and, thereby, th eir function in regulating cytoskeletal assemblies, We have isolated a nother PKC binding protein partial cDNA (clone 45) from a rat fibrobla st library with substantial homology to alpha-adducin. Antisera raised against this expressed sequence recognized a protein of 120 kDa, the reported size of alpha-adducin, on immunoblots of renal proximal tubul e epithelial cell extracts. A 120-kDa protein that cross-reacts with t he clone 45 (alpha-adducin) antisera coprecipitated with 35H immunecom plexes, indicating that alpha-adducin associates with 35H proteins in vivo. Taken together, these results indicate that 35H is a new, widely expressed form of adducin capable of forming heterodimers with alpha- adducin. We propose naming this adducin homologue gamma-adducin.