Functional analysis of CLIP-115 and its binding to microtubules

Cytoplasmic linker proteins (CLIPs) bind to microtubules and are proposed t o link this cytoskeletal network to other intracellular structures. We are interested in CLIP-115, since this protein is enriched in neuronal dendrite s and may operate in the control of brain-specific organelle translocations . Each CLIP monomer is characterized by two microtubule-binding (MTB) motif s, surrounded by basic, serine-rich regions. This head domain is connected to the C-terminal tail through a long coiled-coil structure. The MTB domain s are conserved as a single domain in other proteins involved in microtubul e based transport and dynamics, such as p150(Glued). Here we provide eviden ce that efficient binding of CLIP-115 to microtubules is sensitive to phosp horylation and is not mediated by the conserved MTB domains alone, but requ ires the presence of the basic, serine rich regions in addition to the MTB motifs, In transfected COS-1 cells, CLIP-115 initially accumulates at the d istal ends of microtubules and coincides with CLIP-170, indicating that bot h proteins mark growing microtubule ends. However, when expressed at higher levels, CLIP-115 and -170 affect the microtubule network differently. This might be partly due to the divergent C-termini of the two proteins. We dem onstrate that, similar to CLIP-170, CLIP-115 forms homodimers, which, at le ast in vitro, are linked by disulfide bridges. Cysteine(391) of CLIP-115, h owever, is specific in that it controls the microtubule bundling capacity o f certain mutant CLIP-115 molecules, Therefore, both similar and specific m echanisms appear to regulate the conformation of CLIPs as well as their bin ding to microtubules.