Abstract
GP180 is one of the major transmembrane glycoproteins in mouse T-lymphoma cells. This molecule is an isoform of CD45 and is known to contain an intrinsic protein tyrosine phosphatase (PTPase) activity. Using several complementary biochemical techniques, we have found that fodrin (a spectrin-like protein) is preferentially co-isolated with CD45 (GP180), suggesting that a complex between CD45 (GP180) and the cytoskeleton exists in mouse T-lymphoma cells. Furthermore, we have determined that this CD45 (GP180)-fodrin complex is dissociated by high salt treatment. Using in vitro binding assays, we have shown that CD45 (GP180) binds directly and specifically to fodrin (Kd approximately 1.1 nM) or spectrin (Kd approximately 3.2 nM) in a saturable manner. Additional analyses indicate that a 48-kDa phosphopeptide of CD45 (GP180) contains the fodrin/spectrin-binding domain. Most importantly, the direct binding of fodrin/spectrin to CD45 (GP180) is found to significantly stimulate the PTPase activity of CD45. Enzyme kinetic analysis indicates that fodrin and spectrin increase the Vmax of CD45 (GP180)-mediated dephosphorylation by 7.5 and 3.2-fold, respectively, without significantly changing the Km value. These results strongly suggest that the cytoskeletal proteins, fodrin and spectrin, play an important role in the regulation of the CD45 (GP180) PTPase activity during lymphocyte activation.
Highlights
GP180 isone of the major transmembraneglycopro- regulated during lymphoid development, it is likely that the teins in mouse T-lymphoma cells
Phosphorylation by 7.5 and 3.2-foldr, espectively, bothproteintyrosinekinasesandPTPasesare crucial for without significantly changing the K, value. These results strongly suggest that the cytoskeletal proteins, fodrin and spectrin, play an important role in the regulation of the CD45 (GP180) PTPase activityduring lymphocyte activation
We report that a significant amount of CD45 (GP180) is complexed to fodrin isolated from the plasmamembrane fraction of mouse T-lymphoma cells
Summary
Reagent~-[y-~~P]AT(P>3,000 Ci/mmol) and Iz5I were obtained from Du Pont-New England Nuclear. (GP180)-bound immunobeads (-10-20 ng of protein) were incubated with phosphotyrosyl RCM-lysozyme at a final concentration of p~ in phosphatase assay buffer (200 pl final volume) at 30 "C for 10-. To study the effects of fodrin and spectrin on the PTPaseactivity, CD45 (GP180)-bound immunobeads were preincubated in the presence or absence (control sample) of various concentrations of fodrin or spectrin in the phosphatase assay buffer at 4 "C for 3-4 h with gentle shaking These reaction mixtures were warmed a t 30 "C for 5 min prior to theaddition of phosphotyrosyl RCM-lysozyme. Zyme (-5 PM) is similar to thatreported previously [17], and it did not vary significantly in presence of fodrin or spectrin
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
