Fig. 8.

CaMKII and Munc-18 bind to syntaxin alternatively.A, CaMKII is not bound to Munc-18–syntaxin complex. After recombinant Munc-18 and syntaxin were incubated, the protein complex was immunoprecipitated by anti-syntaxin antibody, and purified CaMKII was added to this complex. Total complex bound to syntaxin was released from GST as described in Materials and Methods.B, CaMKII and Munc-18 alternatively bind to syntaxin in a dose-dependent manner. Both proteins were incubated with the same concentration of immobilized GST–syntaxin 1A at 4°C for 1 hr and eluted with SDS sample buffer. Samples were resolved by 10% SDS-PAGE and immunoblotted. C, CaMKII can bind mutant syntaxin 1A ([1–262]; L165A and E166A; mutant), a forced open form, to which Munc-18 never binds (Dulubova et al., 1999). Control represents normal syntaxin 1A [1–262]. The amount of CaMKII bound to open-form syntaxin was 130.1 ± 4.3% of that bound to wild type, which was significantly increased (n = 4;p < 0.05, t test).