Probing interactions between CLIP-170, EB1, and microtubules.
Cytoplasmic linker protein 170 (CLIP-170) is a microtubule (MT) plus-end tracking protein (+TIP) that dynamically localizes to the MT plus end and regulates MT dynamics. The mechanisms of these activities remain unclear because the CLIP-170-MT interaction is poorly understood, and even less is known about how CLIP-170 and other +TIPs act together as a network. CLIP-170 binds to the acidic C-terminal tail of alpha-tubulin. However, the observation that CLIP-170 has two CAP-Gly (cytoskeleton-associated protein glycine-rich) motifs and multiple serine-rich regions suggests that a single CLIP-170 molecule has multiple tubulin binding sites, and that these sites might bind to multiple parts of the tubulin dimer. Using a combination of chemical cross-linking and mass spectrometry, we find that CLIP-170 binds to both alpha-tubulin and beta-tubulin, and that binding is not limited to the acidic C-terminal tails. We provide evidence that these additional binding sites include the H12 helices of both alpha-tubulin and beta-tubulin and are significant for CLIP-170 activity. Previous work has shown that CLIP-170 binds to end-binding protein 1 (EB1) via the EB1 C-terminus, which mimics the acidic C-terminal tail of tubulin. We find that CLIP-170 can utilize its multiple tubulin binding sites to bind to EB1 and MT simultaneously. These observations help to explain how CLIP-170 can nucleate MTs and alter MT dynamics, and they contribute to understanding the significance and properties of the +TIP network.
Gupta
Kamlesh K
KK
Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556, USA.
Joyce
Michelle V
MV
Slabbekoorn
Aranda R
AR
Zhu
Zhiqing C
ZC
Paulson
Benjamin A
BA
Boggess
Bill
B
Goodson
Holly V
HV
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D003432:Cross-Linking Reagents; D066298:In Vitro Techniques; D008869:Microtubule-Associated Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism*; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D020860:Subtilisin / Q000378:metabolism; D013552:Swine; D014404:Tubulin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D003432:Cross-Linking Reagents; C405804:EB1 microtubule binding proteins; D008869:Microtubule-Associated Proteins; D009363:Neoplasm Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D014404:Tubulin; C076744:cytoplasmic linker protein 170; D020860:Subtilisin