Interaction between FRAP and CLIP-170. (A) Interaction of endogenous FRAP and CLIP-170. HEK293 cell extracts were incubated with a FRAP-specific rabbit polyclonal serum (Imm) or a preimmune serum (Pre). The bound materials were analyzed by western blotting with FRAP (upper) and CLIP-170 (lower) antibodies. T, total input. (B) Interaction of recombinant FRAP and CLIP-170. Left: FLAG-FRAP was immunoprecipitated (IP) from HEK293 cells transiently expressing MYC-CLIP-170 in the presence or absence of FLAG-FRAP. Right: MYC-CLIP-170 was precipitated from lysates of HEK293 cells expressing FLAG-FRAP with or without MYC-CLIP-170. T, total input. (C) FRAP binds to CLIP-170 in vitro. Bacterial recombinant His₆-FRAP(aa 501–930) was incubated with bacterial GST–CLIP-170(aa 1-348) or GST. The bound materials were analyzed by western blotting with an anti-His₆ antibody. T, total input.

Analysis of CLIP-170 phosphorylation. (A) CLIP-170 becomes hyperphosphorylated in the presence of calyculin A. HEK293 cells were treated with and without rapamycin and calyculin A for 30 min. CLIP-170 was resolved on a long 6% SDS polyacrylamide gel and detected by western blotting. (B) CIP-treatment of hyperphosphorylated CLIP-170. Lysates of HEK293 cells treated with calyculin A in the presence and absence of rapamycin were incubated with CIP with or without Na₄P₂O₇. The samples were analyzed by western blotting with a long gel. (C) CLIP-170 is normally hypophosphorylated in vivo. Lysates of HEK293 cells, were incubated with CIP with or without Na₄P₂O₇. CLIP-170 was analyzed by western blotting with a long gel. (D) Metabolic labeling of CLIP-170 with [³²P]orthophosphate. CLIP-170 was isolated from HEK293 cells, metabolically labeled with [³²P]orthophosphate for 3 h and treated without or with rapamycin for 30 min before cell lysis. ³²P-labeled CLIP-170 was separated on an SDS polyacrylamide gel, transferred to a nitrocellulose filter and detected by autoradiography. (E) Tryptic phosphopeptide analysis of in vivo phosphorylated CLIP-170. Samples from (C) were analyzed by 2D tryptic phosphopeptide mapping. Highly reproducible tryptic phosphopeptides were labeled as 1–5. Arrows point to rapamycin-sensitive phosphopeptides. (F) FRAP phosphorylates CLIP-170 in vitro. Wild type and kinase-dead (kd) FLAG-FRAP were incubated with or without CLIP-170 in the presence of [γ-³²P]ATP. The phosphorylated proteins were separated on an SDS polyacrylamide gel, transferred to nitrocellulose filter and detected by autoradiography. (G) FRAP phosphorylates CLIP-170 at the rapamycin-sensitive sites. The 2D tryptic phosphopeptides of in vitro and in vivo phosphorylated CLIP-170 were merged.

Mapping the FRAP-CLIP-170 interaction domains. (A) The N-terminus of CLIP-170 is sufficient to interact with FRAP. Wild type and mutant MYC-CLIP-170 proteins were co-expressed with FLAG-FRAP in HEK293T cells. T, total input; IP, immunoprecipitated materials. (B) The N-terminus of Bik1 interacts with Tor1. Gal4 AD-fused wild type and mutant Bik1 proteins were co-expressed with Gal4 BD-fused Tor1 in a yeast two-hybrid strain. Their interaction was detected by the ability of the yeast strain to grow on a Trp, Leu and His dropout plate containing 5 mM 3-AT. (C) The second HEAT domain of FRAP is responsible for binding to CLIP-170. Wild type and mutant FLAG-FRAP were co-expressed with MYC-CLIP-170 in HEK293T cells. FLAG-FRAP were immunoprecipitated with an anti-FLAG mAb. C, MYC-CLIP-170 was co-transfected with a plasmid vector.

FRAP regulates the ability of CLIP-170 to bind to microtubules. (A) Rapamycin inhibits the binding of CLIP-170 to MTs. Extracts of cells treated with or without rapamycin were incubated with bovine MTs. MT and associated materials were separated on a short gel. MT-bound CLIP-170 was examined by western blotting and bovine tubulin by Coomassie Blue staining. S, supernatant; P, pellet. (B) Rapamycin-sensitive and -insensitive phosphorylation on the MT-binding behavior of CLIP-170. HeLa cells were cultured in the presence or absence of rapamycin before treatment with calyculin A for 15 min. Cell extracts were incubated with bovine MTs. MT-bound materials were separated on a short gel. CLIP-170 was detected by western blotting and bovine tubulin was examined by Coomassie Blue staining. S, supernatant; P, pellet. (C) The effect of rapamycin on CLIP-170 distribution and MT. HeLa cells were cultured in the presence or absence of rapamycin. The cells were stained with CLIP-170- (green) and MT-specific (total MTs, red) antibodies. The highlighted areas were enlarged to show staining details. The arrows show CLIP-170 patches on the growing ends of MTs. (D) A model indicating a possible regulatory mechanism by which CLIP-170 is controlled by phosphorylation.