This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Recently, Rahimi et al. identified an immunoglobulin containing proline rich receptor-1 (IGPR-1) as a novel adhesion receptor involved in tumor cell invasion. They found that the protein SPIN90 is required for IGPR-1 dependent inhibition of cell migration, suggesting that perhaps IGPR-1 alters association of SPIN90 with actin complexes or its localization. SPIN90 also has been shown to be phosphorylated by MAP (ERK1/ERK2), raising a possibility that IGPR-1 may inhibit SPIN90 by interfering with its phosphorylation. Hence, they are interested to evaluate the phosphorylation sites on the SPIN90 after its interaction with IGPR-1. For this purpose, mass spectrometry using bottom-up approach is a method of choice. We performed digestion on the immune-precipitated samples, before analyzing samples by nanoLC-MS/MS on LTQ-Orbitrap (Thermo-Fisher). Preliminary tandem mass spectrometry data performed on SPIN90 by using CID allowed identification of two phosphorylation sites never reported in the literature and another site known to be phosphorylated. However, gel electrophoresis suggested that SPIN90 contained more phosphorylation sites. Hence, further experiments will aim to improve this experiment to increase the sensitivity for the phosphorylated protein (more protein, different phosphate enrichment). ECD or ETD will be used to preserve the phosphorylation, facilitying the identification of the phoshorylation site. To have a better understanding of the mechanism of the interaction between the IGPR-1 and SPIN90, we will also investigate the post-translational modifications on the IGPR-1 (glycosylation, phosphorylation). 1. Rahimi N., Mahoney J.E., Gharahassanlou K.R., Durando M., Meyer R.D., Identification of immunoglobulin containing and proline rich receptor-1 (IGPR-1) as a novel adhesion receptor involved in tumor cell invasion, 2011 2. Lim C.S., Kim S.H., Jung J.G., Kim J.K., Song W.K., Regulation of SPIN90 Phosphorylation and Interaction with Nck by ERK and Cell Adhesion, J. Biol. Chem., 2003, 278, 52, 52116?52123.

Agency
National Institute of Health (NIH)
Institute
National Center for Research Resources (NCRR)
Type
Biotechnology Resource Grants (P41)
Project #
5P41RR010888-15
Application #
8365581
Study Section
Special Emphasis Panel (ZRG1-BCMB-H (40))
Project Start
2011-06-01
Project End
2012-08-09
Budget Start
2011-06-01
Budget End
2012-08-31
Support Year
15
Fiscal Year
2011
Total Cost
$4,616
Indirect Cost
Name
Boston University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
604483045
City
Boston
State
MA
Country
United States
Zip Code
02118
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Wang, Yun Hwa Walter; Meyer, Rosana D; Bondzie, Philip A et al. (2016) IGPR-1 Is Required for Endothelial Cell-Cell Adhesion and Barrier Function. J Mol Biol 428:5019-5033
Steinhorn, Benjamin S; Loscalzo, Joseph; Michel, Thomas (2015) Nitroglycerin and Nitric Oxide--A Rondo of Themes in Cardiovascular Therapeutics. N Engl J Med 373:277-80
Walsh, Erica M; Niu, MengMeng; Bergholz, Johann et al. (2015) Nutlin-3 down-regulates retinoblastoma protein expression and inhibits muscle cell differentiation. Biochem Biophys Res Commun 461:293-9
Théberge, Roger; Dikler, Sergei; Heckendorf, Christian et al. (2015) MALDI-ISD Mass Spectrometry Analysis of Hemoglobin Variants: a Top-Down Approach to the Characterization of Hemoglobinopathies. J Am Soc Mass Spectrom 26:1299-310

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