Sequences in Linker-1 domain of the multidrug resistance associated protein (MRP1 or ABCC1) bind to tubulin and their binding is modulated by phosphorylation
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 (L1{sup MRP1}) interacting proteins. Scanning heptapeptides covering L1{sup MRP1} 126 amino acids (Ile{sup 846}- Leu{sup 972}) 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 L1{sup MRP1} domain [{sup 866}FLRTYAST{sup 867}; {sup 906}SAGKQLQRQLSSS{sup 912}; {sup 925}ISRHHNSTA{sup 927} and {sup 954}AQTGQVKLSVYW{sup 959}] 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-L1{sup MRP1}), we confirmed the peptide scanning approach demonstrating specific binding of tubulin to GST-L1{sup MRP1}. Intriguingly, substitutions of serine residues in L1{sup MRP1} by aspartic acid, but not alanine, abolished its binding to tubulin, suggesting that phosphorylation of Ser{sup 871,} {sup 915,} {sup 930,} {sup and} {sup 961} within L1{sup MRP1} may 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 L1{sup MRP1} domain. - Highlights: • This study shows direct binding between MRP1 Linker 1 (L1{sup MRP1}) domain and α- and β-tubulin subunits, using a scanning peptide approach. • Four short and dispersed sequences in L1{sup MRP1} domain interact with α/β-tubulin subunits. • Each sequence in L1{sup MRP1}-tubulin binding domain contains predicted protein kinase C/A site. • Substituting 3 of 4 serine with aspartic acid, but not alanine, in L1{sup MRP1} binding domains inhibit their binding to tubulin subunits.
- OSTI ID:
- 22696792
- Journal Information:
- Biochemical and Biophysical Research Communications, Vol. 482, Issue 4; Other Information: Copyright (c) 2016 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved.; Country of input: International Atomic Energy Agency (IAEA); ISSN 0006-291X
- Country of Publication:
- United States
- Language:
- English
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