title: Sequences in Linker-1 domain of the multidrug resistance associated protein (MRP1 or ABCC1) bind to tubulin and their binding is modulated by phosphorylation.
abstract: The multidrug resistant associated protein 1 (or MRP1, ABCC1) encodes two cytoplasmic linker domains (L0 and L1) composed of highly charged sequences with multiple protein kinase A/C phosphorylation sites. In this report we made use of the scanning peptide approach to identify MRP1 linker L1 (L1MRP1) interacting proteins. Scanning heptapeptides covering L1MRP1126 amino acids (Ile846- Leu972) were synthesized and used in pull-down assays to isolate proteins from cell extracts (human multidrug resistant SCLC cell line; H69/AR). The results show four high affinity binding sequences in L1MRP1domain [866FLRTYAST867;906SAGKQLQRQLSSS912;925ISRHHNSTA927and954AQTGQVKLSVYW959] that bound ∼55 kDa and 110 kDa polypeptides. The latter polypeptides were identified by mass spectrometry as α- and β-tubulin monomers and dimers. Western blotting with monoclonal antibodies to α- and β-tubulin proteins confirmed the mass-spectrometry results. Moreover, using recombinant full-length GST-Linker 1 fusion polypeptide (GST-L1MRP1), we confirmed the peptide scanning approach demonstrating specific binding of tubulin to GST-L1MRP1. Intriguingly, substitutions of serine residues in L1MRP1by aspartic acid, but not alanine, abolished its binding to tubulin, suggesting that phosphorylation of Ser871, 915, 930, and 961within L1MRP1may modulate its binding to tubulin. Taken together, the results of this study suggest possible interaction between MRP1 and tubulin that is modulated by phosphorylation of specific sequences in the L1MRP1domain.
authors.affiliation: Institute of Parasitology, McGill University, Quebec, Canada.
authors.forename: Raghuram
authors.initials: R
authors.lastname: Ambadipudi
authors.affiliation: Institute of Parasitology, McGill University, Quebec, Canada. Electronic address: elias.georges@mcgill.ca.
authors.forename: Elias
authors.initials: E
authors.lastname: Georges
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D045744:Cell Line, Tumor; D006801:Humans; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D060066:Protein Interaction Maps; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D014404:Tubulin / Q000737:chemistry / Q000378:metabolism*
chemical_list: D027425:ATP-Binding Cassette, Sub-Family C Proteins; D021122:Protein Subunits; D014404:Tubulin; C421109:multidrug resistance-associated protein 1
keywords: ABCC1; Linker 1 domain; MRP1; Phosphorylation; Protein interactions; Tubulin