Phosphorylation controls autoinhibition of cytoplasmic linker protein-170.
Cytoplasmic linker protein (CLIP)-170 is a microtubule (MT) plus-end-tracking protein that regulates MT dynamics and links MT plus ends to different intracellular structures. We have shown previously that intramolecular association between the N and C termini results in autoinhibition of CLIP-170, thus altering its binding to MTs and the dynactin subunit p150(Glued) (J. Cell Biol. 2004: 166, 1003-1014). In this study, we demonstrate that conformational changes in CLIP-170 are regulated by phosphorylation that enhances the affinity between the N- and C-terminal domains. By using site-directed mutagenesis and phosphoproteomic analysis, we mapped the phosphorylation sites in the third serine-rich region of CLIP-170. A phosphorylation-deficient mutant of CLIP-170 displays an "open" conformation and a higher binding affinity for growing MT ends and p150(Glued) as compared with nonmutated protein, whereas a phosphomimetic mutant confined to the "folded back" conformation shows decreased MT association and does not interact with p150(Glued). We conclude that phosphorylation regulates CLIP-170 conformational changes resulting in its autoinhibition.
Department of Cell and Molecular Biology, Northwestern University Medical School, Chicago, IL 60611, USA.
Ho-Sup
HS
Lee
Yulia A
YA
Komarova
Elena S
ES
Nadezhdina
Rana
R
Anjum
John G
JG
Peloquin
Joseph M
JM
Schober
Oana
O
Danciu
Jeffrey
J
van Haren
Niels
N
Galjart
Steven P
SP
Gygi
Anna
A
Akhmanova
Gary G
GG
Borisy
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004398:Dyneins / Q000378:metabolism; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D008870:Microtubules / Q000187:drug effects / Q000378:metabolism; D008954:Models, Biological; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009363:Neoplasm Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D019319:Okadaic Acid / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D010768:Phosphoserine / Q000378:metabolism; D017510:Protein Folding / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects; D020543:Proteome / Q000378:metabolism
D008869:Microtubule-Associated Proteins; D009363:Neoplasm Proteins; D020543:Proteome; C076744:cytoplasmic linker protein 170; D010768:Phosphoserine; D019319:Okadaic Acid; D017868:Cyclic AMP-Dependent Protein Kinases; D004398:Dyneins