Adducin Regulation: DEFINITION OF THE CALMODULIN-BINDING DOMAIN AND SITES OF PHOSPHORYLATION BY PROTEIN KINASES A AND C

Yoichiro Matsuoka,Christine A Hughes,Vann Bennett

doi:10.1074/jbc.271.41.25157

Yoichiro Matsuoka, Christine A Hughes + Show 1 more

Open Access

https://doi.org/10.1074/jbc.271.41.25157

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Abstract

Adducin promotes association of spectrin with actin and caps the fast growing end of actin filaments. Adducin contains N-terminal core, neck, and C-terminal tail domains, is a substrate for protein kinases A (PKA) and C (PKC), and binds to Ca2+/calmodulin. Ser-726 and Ser-713 in the C-terminal MARCKS-related domains of alpha- and beta-adducin, respectively, were identified as the major phosphorylation sites common for PKA and PKC. PKA, in addition, phosphorylated alpha-adducin at Ser-408, -436, and -481 in the neck domain. Phosphorylation by PKA, but not PKC, reduced the affinity of adducin for spectrin-F-actin complexes as well as the activity of adducin in promoting binding of spectrin to F-actin. The myristoylated alanine-rich protein kinase C substrate-related domain of beta-adducin was identified as the dominant Ca2+-dependent calmodulin-binding site. Calmodulin-binding was inhibited by phosphorylation of beta-adducin and of a MARCKS-related domain peptide by PKA and PKC. Calmodulin in turn inhibited the rate, but not the extent, of phosphorylation of beta-adducin, but not alpha-adducin, by PKA and that of each subunit by PKC. These findings suggest a complex reciprocal relationship between regulation of adducin function by calmodulin binding and phosphorylation by PKA and PKC.

Highlights

Adducin promotes association of spectrin with actin and caps the fast growing end of actin filaments
Calmodulin in turn inhibited the rate, but not the extent, of phosphorylation of ␤-adducin, but not ␣-adducin, by protein kinases A (PKA) and that of each subunit by protein kinase C (PKC). These findings suggest a complex reciprocal relationship between regulation of adducin function by calmodulin binding and phosphorylation by PKA and PKC
Functional consequences of phosphorylation reported here include modulation of adducin interactions with spectrin and actin by PKA and inhibition of calmodulin binding by PKA and PKC

Summary

EXPERIMENTAL PROCEDURES

Purification of Proteins—Rabbit muscle actin, bovine brain spectrin, bovine brain calmodulin, and erythrocyte adducin were isolated essentially as described [14]. Recombinant human ␣- (residues 430 –737) and ␤- (residues 409 –726) adducin neck/tail constructs were expressed as soluble proteins and purified as described [14]. Modification of Phosphoserine Residue—The purified radioactive fragments (1–3 nmol) were treated with 100 ␮l of solution consisting of 9.9 ␮l of ethanethiol, 33.1 ␮l of water, 33.1 ␮l of dimethyl sulfoxide, 13.2 ␮l of ethanol, and 10.7 ␮l of 5 N NaOH at 50 °C for 1 h as described by Meyer et al [23] In this condition, a phosphoserine residue is converted into S-ethylcysteine and can be detected by amino acid sequence analysis. The excitation wavelength was 340 nm, and emission was monitored at 480 nm with a spectrofluorophotometer. 125I-Azidocalmodulin binding was determined as described [2]

RESULTS

DISCUSSION

Methods