Interactions of the Pleckstrin Homology Domains of M-RIP (p116Rip) with F-actin

M-RIP has been shown to interact with actin, myosin, RhoA and the targeting subunit of myosin phosphatase, and has been proposed to be a scaffolding protein that anchors RhoA and Rho kinase onto myosin phosphatase and the actomyosin cytoskeleton. The N-terminal portion of M-RIP has 2 pleckstrin homology domains at residues 44–152 and 387–484 (PH1 and PH2, respectively). Mulder et al. (J. Biol. Chem. 278, 27216–23, 2003) showed that the actin-binding activity of M-RIP resides in its N-terminal portion, and characterized the interaction between PH1 and F-actin, but did not investigate the role of PH2 in M-RIP's actin binding activity. In this work we examined the contributions of PH1 and PH2 to the M-RIP-F-actin interaction by, first, constructing 3 deletion mutants of M-RIP: M-RIP(1-386) (PH1), M-RIP(146-492) (PH2) and M-RIP(1-492) (PH1 and PH2). Co-sedimentation experiments with F-actin were then carried out at low or high speeds to sediment M-RIP-bound to F-actin bundles or filaments, respectively. The following results were obtained: 1) The extent of bundle formation increases in the order M-RIP(1-386) < M-RIP(146-492) < M-RIP(1-492). 2) The extent of total actin binding increases in the order M-RIP(1-386) < M-RIP(146-492) < M-RIP(1-492). Sequence homology analysis revealed that certain basic residues known to be important for actin binding in other PH domain-containing proteins (Yao et al. J. Biol. Chem. 274, 19752–61, 1999) are present in PH2. Mutagenesis of three such residues, Lys404, 405 and 396, into Ala eliminated the binding between M-RIP(1-492) and F-actin. Taken together our results show that the binding of M-RIP to F-actin is mediated primarily via its PH2 domain, in particular via ionic interactions between certain basic residues in PH2 and acidic residues in actin. (Supported by NIH AR41637 and AR49066).