The macromolecular assembly of protein serine/threonine phosphatasekinase complexes mechanistically enhances the speed and fidelity of cell signaling events. These signaling modules allow for acute regulation of substrates common to both enzymes as well as intramolecular regulation of kinase activity by the associated phosphatase or vice versa. Protein serine/threonine phosphatase 2A (PP2A) interacts with calcium/calmodulin-dependent protein kinase IV (CaMKIV) and negatively regulates the activity of the kinase. A phospho-Thr200-CaMKIV antibody was utilized to monitor ionomycin-induced signal output of endogenous CaMKIVPP2A complexes. The PP2A canonical regulatory Balpha and Bdelta subunits recruit the catalytic subunit of PP2A (PP2AC) to the kinase but do not promote dephosphorylation of the activating phospho-Thr200 site. Balpha and Bdelta subunits also recruit PP2AC to the serine/threonine kinase Raf1. In contrast to CaMKIV, PP2A positively regulates Raf1-MEK-ERK signaling by dephosphorylating the inhibitory phospho-Ser259-Raf1 residue. To better understand the regulatory mechanisms controlling ABalpha/deltaC-Raf1 interactions in response to stimuli, we coupled Reversible Cross-Link Immuno-Precipitation (ReCLIP) with mass spectrometry. Tandem affinity-purified ABdeltaCRaf1 complexes were isolated from cells treated with a chemical crosslinking reagent to stabilize transient protein-protein interactions. Proteomic analysis of the PP2ARaf1 complex identified several putative interacting proteins that may play a role in the regulation of this phosphatasekinase complex. Raf1 signaling is inactivated following the dephosphorylation of phospho-Ser338 by protein serine/threonine phosphatase 5 (PP5). We monitored the binding of PP2A and PP5 to Raf1 using co-immunoprecipitation assays and found that both phosphatases concurrently associate with Raf1, which acts as the scaffold in this multiprotein complex. We also identify several novel PP5kinase complexes whose stable interactions are facilitated by HSP90. Notably, PP5 interacts with multiple ERKs. Our studies support a novel role for PP5ERK complexes in regulating the feedback phosphorylation of Raf1 at Ser-289, Ser-296, and/or Ser-301 and show that these PP5ERK complexes are regulated by small G proteins. Whereas constitutively active Rac1 promotes the assembly of PP5ERK1/2 complexes, oncogenic HRas has no effect on PP5-ERK1 binding but selectively decreases the PP5-ERK2 interaction, in a manner that is independent of PP5 and MEK activity, yet paradoxically requires ERK2 activity.
| Identifer | oai:union.ndltd.org:VANDERBILT/oai:VANDERBILTETD:etd-03242014-134955 |
| Date | 14 April 2014 |
| Creators | Mazalouskas, Matthew David |
| Contributors | Vsevolod Gurevich, Brian Wadzinski, Albert Reynolds, BethAnn McLaughlin, Claus Schneider |
| Publisher | VANDERBILT |
| Source Sets | Vanderbilt University Theses |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.vanderbilt.edu/available/etd-03242014-134955/ |
| Rights | restrictone, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to Vanderbilt University or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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