35H, a Sequence Isolated as a Protein Kinase C Binding Protein, Is a Novel Member of the Adducin Family

doi:10.1074/jbc.270.43.25534

35H, a Sequence Isolated as a Protein Kinase C Binding Protein, Is a Novel Member of the Adducin Family (*)

From the W. Alton Jones Cell Science Center, Inc., Lake Placid, New York 12946-1099

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Abstract

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 α- and β-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 α- and β-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 α-adducin. Antisera raised against this expressed sequence recognized a protein of 120 kDa, the reported size of α-adducin, on immunoblots of renal proximal tubule epithelial cell extracts. A 120-kDa protein that cross-reacts with the clone 45 (α-adducin) antisera coprecipitated with 35H immunecomplexes, indicating that α-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 α-adducin. We propose naming this adducin homologue -adducin.

Footnotes

↵* This work was supported by NIH Grants ES05670 (to J. L. S. and S. J.), CA53841 and GM50152 (to S. J.), Council for Tobacco Research Grant 2375A (to S. J.), and American Cancer Society Grant CN-105A (to S. J.). Portions of this work were submitted in partial fulfillment of requirements for the Ph.D. degree awarded to L. D. from Clarkson University (Potsdam, NY). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) U35775[GenBank].
↵¹ Abbreviations used are:

PKC

protein kinase C

CSK

cytoskeleton or cytoskeletal

MARCKS

myristoylated alanine-rich protein kinase C substrate

MSB

microtubule stabilization buffer

PDBu

phorbol 12,13-dibutyrate

RPTE

renal proximal tubule epithelial cells

PIPES

1,4-piperazinediethanesulfonic acid

kb

kilobase pair(s)

PS

phosphatidylserine.
↵²C. Chapline, personal communication.
- Received May 31, 1995.
- Revision received July 20, 1995.