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) binding protein from a rat kidney cDNA library and demonstrated that it is a PKC substrate in vitro (Chapline, C., Ramsay, K., Klauck, T., and 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 sequence analysis, and Northern analysis indicate that 35H is a unique cDNA related to alpha-and beta-adducins. Antisera prepared to the 35H bacterial fusion protein recognized two polypeptides of 80 and 90 kDa on immunoblots of kidney homogenates and cultured renal proximal tubule epithelial cell extracts. The 35H-related proteins were similar to alpha- and beta-adducins in that they were preferentially recovered in the Triton X-100-insoluble (cytoskeletal, CSK) fraction of cell extracts and were predominantly localized to cell borders. Phorbol esters stimulated phosphorylation of CSK 35H proteins, thus emphasizing that sequences isolated according to PKC binding activity in vitro are also PKC substrates in vivo. The phosphorylated forms of the 35H proteins were preferentially recovered in the soluble fraction, thus demonstrating that phosphorylation regulates their CSK association and, thereby, their function in regulating cytoskeletal assemblies. We have isolated another PKC binding protein partial cDNA (clone 45) from a rat fibroblast 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 tubule epithelial cell extracts. A 120-kDa protein that cross-reacts with the clone 45 (alpha-adducin) antisera coprecipitated with 35H immunecomplexes, 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.
Dong
L
L
W. Alton Jones Cell Science Center, Inc., Lake Placid, New York 12946-1099, USA.
Chapline
C
C
Mousseau
B
B
Fowler
L
L
Ramsay
K
K
Stevens
J L
JL
Jaken
S
S
D000595:Amino Acid Sequence; D000818:Animals; D002148:Calmodulin-Binding Proteins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism*; D002451:Cell Compartmentation / Q000187:drug effects; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015151:Immunoblotting; D007668:Kidney / Q000737:chemistry; D007687:Kidney Tubules, Proximal / Q000378:metabolism; D008297:Male; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D010766:Phosphorylation; D011233:Precipitin Tests; D011485:Protein Binding; D011493:Protein Kinase C; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D011916:Rats, Inbred F344; D011993:Recombinant Fusion Proteins / Q000276:immunology; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014018:Tissue Distribution; D000099291:Adducins
D002148:Calmodulin-Binding Proteins; D018076:DNA, Complementary; D015240:Phorbol 12,13-Dibutyrate; D011493:Protein Kinase C; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D000099291:Adducins