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J. Biol. Chem., Vol. 279, Issue 4, 3068-3077, January 23, 2004

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CARMIL Is a Bona Fide Capping Protein Interactant^*

Kirsten Remmert, Thomas E. Olszewski, M. Blair Bowers, Mariana Dimitrova, Ann Ginsburg, and John A. Hammer, III¶

From the Laboratory of Cell Biology and Laboratory of Biochemistry, NHLBI, National Institutes of Health, Bethesda, Maryland 20892

CARMIL, also known as Acan 125, is a multidomain protein that was originally identified on the basis of its interaction with the Src homology 3 (SH3) domain of type I myosins from Acanthamoeba. In a subsequent study of CARMIL from Dictyostelium, pull-down assays indicated that the protein also bound capping protein and the Arp2/3 complex. Here we present biochemical evidence that Acanthamoeba CARMIL interacts tightly with capping protein. In biochemical preparations, CARMIL copurified extensively with two polypeptides that were shown by microsequencing to be the - and -subunits of Acanthamoeba capping protein. The complex between CARMIL and capping protein, which is readily demonstratable by chemical cross-linking, can be completely dissociated by size exclusion chromatography at pH 5.4. Analytical ultracentrifugation, surface plasmon resonance and SH3 domain pull-down assays indicate that the dissociation constant of capping protein for CARMIL is 0.4 µM or lower. Using CARMIL fusion proteins, the binding site for capping protein was shown to reside within the carboxyl-terminal, 200 residue, proline-rich domain of CARMIL. Finally, chemical cross-linking, analytical ultracentrifugation, and rotary shadowed electron microscopy revealed that CARMIL is asymmetric and that it exists in a monomer dimer equilibrium with an association constant of 1.0 x 10⁶ M^-1. Together, these results indicate that CARMIL self-associates and interacts with capping protein with affinities that, given the cellular concentrations of the proteins (1 and 2 µM for capping protein and CARMIL, respectively), indicate that both activities should be physiologically relevant.

Received for publication, August 11, 2003 , and in revised form, October 23, 2003.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Laboratory of Cell Biology, NHLBI, Bldg. 50, Rm. 2523, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892-8017. Tel.: 301-496-1001; Fax: 301-402-1519; E-mail: hammerj{at}nhlbi.nih.gov.

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