The Regulation of CLIP-170-Microtubule Binding

CLIP-170 is a microtubule (MT) binding protein that binds preferentially to the MT plus-end via its CAP-Gly domain. CLIP-170 is a highly conserved protein that plays multiple roles in the cell. Initially identified as a linker between membranes and the microtubule cytoskeleton (Pierre et al., 1992), more recently CLIP-170 has been shown to dynamically track the growing end (plus-end) of MTs in vivo (Perez et al., 1999) and enhance MT polymerization both in vitro and in vivo (Diamantopoulos et al., 1999). The CLIP-170-MT interaction is highly dynamic in vivo and precipitates the question of how CLIP-170 tracks growing MT plus-ends and then suddenly disappears when the MT stops growing.

One prediction, that the interaction is tightly regulated, is based on several observations. Previous work has shown that the CLIP-170-MT interaction can be manipulated in lysates using the phosphatase inhibitor okadaic acid and potato acid phosphatase (Rickard and Kreis, 1991). The dynamic interaction between p150glued, the MT binding component of dynactin, and MTs is modulated by phosphorylation (Vaughan et al., 2002).

Finally, the kinetics of CLIP-170 binding and release appear to be very different in vivo and in vitro (Folker et al., 2005). These ideas lead to the possibility that phosphorylation could be important in CLIP-170-MT associations. To test this hypothesis and help define the mechanism of CLIP-170 regulation, I have investigated the role of phosphorylation in CLIP-170-MT interactions. More specifically, 6 amino acids were identified as phosphorylated in the CLIP-170 fragment H2. These sites were mutated and analyzed with several mutants showing altered MT binding- H1 S195A, H1 S204E and H1 6E. Taken as a whole, these results imply a plus-end tracking mechanism where CLIP-170 binds the MT plus end in a phosphorylated state and subsequent phosphorylations cause it to fall off the MT.