Adducin Regulation

doi:10.1074/jbc.271.41.25157

Adducin Regulation

DEFINITION OF THE CALMODULIN-BINDING DOMAIN AND SITES OF PHOSPHORYLATION BY PROTEIN KINASES A AND C*

From the Howard Hughes Medical Institute and Departments of Cell Biology and Biochemistry, Duke University Medical Center, Durham, North Carolina 27710

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 α- and β-adducin, respectively, were identified as the major phosphorylation sites common for PKA and PKC. PKA, in addition, phosphorylated α-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 β-adducin was identified as the dominant Ca²⁺-dependent calmodulin-binding site. Calmodulin-binding was inhibited by phosphorylation of β-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 β-adducin, but not α-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.

Footnotes

↵* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

PKC

protein kinase C

PKA

cAMP-dependent protein kinase

MARCKS

myristoylated alanine-rich protein kinase C substrate

DTT

dithiothreitol

HPLC

high pressure liquid chromatography

MOPS

4-morpholinepropanesulfonic acid

CaM-DANS

5-dimethylaminonaphthalene-1-sulfonyl-(dansyl)-calmodulin.
↵² P. J. Blackshear, unpublished data.
↵³ C. A. Hughes, unpublished data.
↵⁴ Y. Matsuoka, unpublished data.
- Received May 25, 1996.
- Revision received July 15, 1996.