Microtubules (MT) are highly dynamic, polarized cytoskeleton filaments that are responsible for multiple kinds of movements and transport in all eukaryotic cells. A large number of proteins bind to MT with many different function. A subset of binding proteins associates specifically with the plus ends of MTs. Proteins including dynactin, EB1 and CLIP170 display a behavior known as "tip-tracking" when they are expressed as GFP or RFP fusion proteins (Vaughan, 2004). Recent work on p150^Glued, a subunit of dynactin, suggests that tip-tracking reflects a dynamic association with MT which is regulated by phosphorylation. We have shown that members of the PKA family are involved in regulation of p150^Glued to MT, however other kinases have also been implicated. The mammalian target of rapamycin (mTOR) has been identified recently as another potential kinase that regulates the MT tip-tracking protein CLIP170 (Choi et al., 2002). mTOR modulates a large number of major cellular processes, including proliferation and cell growth. We have assessed the role of mTOR phosphorylation on p150^Glued and EB1 by immunofluorescence and live-cell imaging of cells expressing GFP-p150^Glued or EB1-GFP after treatment with rapamycin, and imaging of cells over-expressing wild-type, kinase-dead and rapamycin-resistant mTOR mutants. Our findings reveal that mTOR has a differential effect on p150^Glued and EB1. Based on the results of rapamycin treatment, it appears that mTOR activity promotes the binding of p150^Glued to microtubules (in contrast to PKA) but directs the release of EB1 from MTs. 2-D gel analysis of control and rapamycin-treated COS-7 cell extracts revealed that inhibition of mTOR with rapamycin results in an increase in the p150^Glued phosphorylation complexity, suggesting that the effect on p150^Glued might to be indirect. We also we performed in-vitro microtubule-binding assays using recombinant p150^Glued (1-330) in the presence of extracts from COS-7 cells treated or not with rapamycin. Clearly rapamycin-treated extracts decreased the affinity of p150^Glued for MTs (via p150^Glued phosphorylation) consistent with our imaging results. In summary, our findings suggest that microtubule tip-tracking proteins are regulated by multiple kinase pathways.