Figure 5. Association of the CaM-IP2 complex with CaM is dis- rupted in DRC mutants. (A) Silver-stained gel of immunoprecipitates using either anti-CaM or anti–CaM-IP2 antibodies and axonemal extracts isolated from wild-type (wt), spokeless (pf14), or DRC mu- tant (pf2 and pf3) axonemes. Compared with wild-type and pf14 precipitates, the CaM-IP2 complex is substantially reduced in pre- cipitates of pf2 and pf3 extracts using the anti-CaM antibodies. However, immunoprecipitation using the anti–CaM-IP2 antibodies indicates that relatively equal amounts of CaM-IP2, -IP3, and -IP4 (asterisks) are precipitated in wild-type, pf2, and pf3 axonemes. (B) Western blot of isolated axonemes using anti-CaM antibodies. The pf2 and pf3 mutant axonemes have wild-type levels of CaM. These combined results indicate that association of the CaM-IP2 complex with CaM is disrupted in DRC mutants. central apparatus and outer dynein arms (pf18pf28) or radial spokes and outer dynein arms (pf14pf28) have shorter length flagella than wild type, and many of the isolated axonemes fail to undergo microtubule sliding in this assay. However, for those pf18pf28 axonemes in which microtubule sliding occurred, velocities doubled upon addition of the CaM-IP2 antibodies (Fig. 6 C). These velocities are significantly lower than those of pf28 (P < 0.001 by t test). The CaM-IP2 antibodies had no ef- fect on the sliding velocity of pf14pf28 axonemes. These results indicate that the outer dynein arms may be one of but not the exclusive targets of regulation by the CaM complex. Therefore, we also compared dynein activity in double mutants lacking various inner dynein arm subforms. Addition of the CaM-IP2 antibody restored dynein activ- ity in mutant axonemes lacking the central apparatus and the I2 inner arms (pf18ida4; Fig. 6 D; Piperno et al., 1992), which are also known as fast protein liquid chromatography fractions a, c, and d (Kagami and Kamiya, 1992; Porter et al., 1992). The CaM-IP2 antibodies increased sliding velocities of pf18ida4 axonemes to levels not significantly different (P > 0.22 by t test) from those of pf18 axonemes incubated with CaM-IP2 antibody. These results suggest that the I2 inner arms are not the targets of regulation by this complex. The pf30 and ida1 mutants lack the inner arm I1 or fraction f heavy chains (Piperno et al., 1990; Kagami and Kamiya, 1992; Porter et al., 1992). Addition of the CaM-IP2 antibodies to axonemes isolated from the pf14pf30 mutant that lack the spokes and the I1 inner dynein arm failed to increase dynein activity (Fig. 6 D). These results indicate that I1 is a target for regulation by this complex. We previously reported that double mutants lacking I1 and the central apparatus (pf18ida1) have micro- tubule sliding velocities that are significantly greater than those of pf18 axonemes (P < 0.001 by t test; Smith, 2002b). The loss of I1 from central pairless mutants partially relieves the inhibition of microtubule sliding in central apparatus–defective mutants. However, sliding velocities of pf18ida1 are also significantly lower than those of pf18 axonemes exposed to the CaM-IP2 anti- bodies (P < 0.001 by t test; Fig. 6 D). Addition of the CaM-IP2 antibodies to pf18ida1 mutant axonemes failed to increase dynein activity to wild-type levels. These combined results support the hypothesis that the CaM-IP2, -IP3, and -IP4 complex plays a role in modulating the activity of inner dynein arm I1. Previous studies have indicated that reduced sliding ve- locities in central pairless and radial spokeless mutants is corre- lated with the phosphorylation state of the IC138 intermediate chain of the I1 inner dynein arm (Habermacher and Sale, 1996, 1997; King and Dutcher, 1997; Yang and Sale, 2000; Hendrickson et al., 2004). Reducing the phosphorylation of IC138 using ki- nase inhibitors results in increased sliding velocities in these mutants (Habermacher and Sale, 1997; Yang and Sale, 2000; Smith, 2002b). Therefore, we hypothesized that addition of the CaM-IP2 antibodies to mutant axonemes may result in reduced IC138 phosphorylation and, therefore, increased dynein activity. Using antibodies generated against IC138, we performed West- ern blots of isolated axonemes in the presence or absence of CaM-IP2 antibodies (Fig. 7). Phosphorylated forms of IC138 have been detected on Western blots of radial spoke– and cen- tral apparatus–defective axonemes as slower migrating species that are not present when samples are treated with phosphatase (Hendrickson et al., 2004). We do not detect any decrease in the overall phosphorylation state of IC138 in either radial spokeless (pf14) or central pairless (pf18) axonemes upon the addition of anti–CaM-IP2 antibodies. These results suggest that the CaM-IP2 antibodies do not increase dynein activity by affecting the phosphorylation state of I1 IC138. Alternatively, the phos- phorylation state of specific residues may be affected that can- not be detected by Western blotting. Discussion Our previous studies demonstrated that calcium control of motility involves the regulation of dynein-driven microtubule sliding and that CaM is a key axonemal calcium sensor (Smith, 2002a,b; Wargo et al., 2004). Using a combination of biochem- ical and functional approaches, we have identified a protein com- plex that exhibits calcium-sensitive CaM binding, localizes to 520 JCB • VOLUME 179 • NUMBER 3 • 2007