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

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 ␣-and ␤-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 ␣-and ␤-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 ␣-adducin. Antisera raised against this expressed sequence recognized a protein of 120 kDa, the reported size of ␣-adducin, on immunoblots of renal proximal tubule epithelial cell extracts. A 120-kDa protein that cross-reacts with the clone 45 (␣-adducin) antisera coprecipitated with 35H immunecomplexes, indicating that ␣-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 ␣-adducin. We propose naming this adducin homologue ␥-adducin.Protein kinase Cs are a heterogeneous group of phospholipiddependent kinases important for cell growth and differentiated functions (reviewed in Ref. 1). The family can be divided into three categories based on enzymatic properties. The conventional or Group A PKCs 1 are calcium-dependent kinases whose activities are stimulated by diacylglycerol or phorbol esters. The novel or Group B PKCs are calcium-independent but still diacylglycerol-stimulatable. The atypical or Group C PKCs are calcium-and diacylglycerol-independent. Most cells express more than one type of PKC, which implies that PKCs have unique rather than overlapping functions.Activation of Group A and B PKCs is regulated by receptormediated production of diacylglycerol through phospholipase C or D pathways (1). In many cases, activation correlates with PKC redistribution from soluble to particulate fractions. However, in other cases, evidence for activation in the absence of measurable translocation has been noted (2-5). The correlation ...

Increased protein kinase Cδ in mammary tumor cells: relationship to transformation and metastatic progression

“…Mouse anti-HA monoclonal (clone 12CA5) antibody was from Boehringer-Mannheim (Indianapolis, IN, USA). Antibodies for a-and g-adducin have been described (Dong et al, 1995). Rabbit anti-pSer660-adducin antiserum was raised to a phosphopepetide containing the PKC phosphorylation site in a, b and g adducins.…”

“…Adducin phosphorylated by PKC was monitored with a phosphorylation state selective antibody raised against a g-adducin phosphopeptide containing the PKC phosphorylation site at ser660 (Fowler et al, 1998a,b). This antibody recognizes g-adducin phosphorylated at ser660 and aadducin phosphorylated at the homologous site at ser729 (Fowler et al, 1998a;Dong et al, 1995). Immuno¯uorescence studies demonstrated two pools of pSer660-adducin, perinuclear cytoplasmic and peripheral.…”

Adducin Promotes Micrometer-Scale Organization of β2-Spectrin in Lateral Membranes of Bronchial Epithelial Cells

“…Adducins are a family of three closely related polypeptides encoded by distinct genes (␣-, ␤-, and ␥-adducins; Joshi et al, 1991;Dong et al, 1995). ␣-Adducin forms heterodimers and tetramers with ␤-adducin and most likely ␥-adducin (Dong et al, 1995;Hughes and Bennett, 1995).…”

“…Considering adducin forms ␣-␤ and likely ␣-␥ heterotetramers (Hughes and Bennett, 1995;Dong et al, 1995), we chose to target ␣-adducin for shRNA depletion. This strategy eliminates the possibility of compensation through up-regulation of ␤-adducin in HBE cells, which is a concern based on the finding that ␥-adducin is up-regulated in RBCs of the ␤-adducin knockout mouse (Gilligan et al, 1999).…”

Adducin‐1 is essential for spindle pole integrity through its interaction with TPX2

“…The tail domain is composed of a carboxyl‐terminal stretch of 22 residues with homology to the myristoylated alanine‐rich C kinase substrate (MARCKS) domain 29, 32. Adducin interacts with spectrin, calmodulin, and F‐actin through the MARCKS‐related motif 29, 32, 33. Phosphorylation in this motif by protein kinase C or protein kinase A decreases its ability to bind spectrin and F‐actin 34, 35.…”