Signal transduction is a term used to describe the series of events that culminates in a cellular response to an extracellular signal or environmental stimuli. From the engagement of cell surface receptors to the activation of protein kinase cascades, this process involves a variety of molecular participants and regulates numerous cellular processes including cell survival, vesicular trafficking, cell differentiation, mRNA dynamics, and cellular proliferation.
Cellular phosphorylation is regulated by two enzyme superfamilies - kinases and phosphatases and utilization of this post translational modification is at the heart of signal transduction. Activation of kinase and phosphatase cascades leads to strategic changes in intracellular phosphorylation and subsequently the function of target molecules allowing the cell and organism to respond appropriately to its surroundings. Disruption of signaling integrity invariably leads to abnormal phosphorylation, a consequence that often causes or promotes many human diseases.
Our laboratory integrates biological mass spectrometry and other proteomic techniques with traditional biochemical and cell biology methods to decipher the mechanistic details regulating poorly understood kinases and phosphatases predicted to function in processes such as cellular proliferation, cell survival, and neuromuscular signaling.
Current projects include:
I. Molecular mechanisms regulating the Myotubularin lipid phosphatase MTMR2 mutated in the neuromuscular disorder Charcot-Marie Tooth disease - Mutations in the MTMR2 gene has been shown to cause Charcot-Marie Tooth disease 4B1 (CMT4B1). CMT4B1 is a demyelinating neuropathy characterized by abnormally folded myelin sheaths causing inadequate nerve conduction to the muscles eventually leading to muscle weakness and atrophy. Using mass spectrometry, we mapped a stable phosphorylation site close to the PH-GRAM domain of MTMR2 that serves as a master regulator of MTMR2 endosome targeting. Our lab discovered that phosphorylation at Ser58 is mediated by ERK1/2 in a negative feedback mechanism and markedly decreases MTMR2 localization to early endosomes. In contrast, a phosphorylation-deficient MTMR2 strongly localizes to early endocytic structures, depletes its physiological substrate PI3P, halts endosome maturation, and enhances growth factor signaling. This was the first report documenting a role for reversible phosphorylation regulating MTMR2 activities and reveals a critical mechanism for controlling MTMR2’s access to its physiological substrate that potently controls endosomal dynamics. We are currently investigating the role of MTMR2 phosphorylation during myelination and Schwann cell differentiation. We have also initiated a mass spectrometry based method aimed at identifying target proteins that are affected by MTMR2 enzymatic activity in disease-related cells. We have identified and verified one candidate (RME8) which has a characterized role in vesicular transport and recycling receptors to the cell surface. We now plan to further define the proteome that interacts with the lipid substrates of MTMR2.
II. Functional characterization of hYVH1: a unique dual specificity phosphatase that regulates cell survival and ribosome biogenesis - Our laboratory discovered that the evolutionary conserved dual specificity phosphatase hYVH1 (also known as DUSP12) is a potent cell survival enzyme and a novel regulator of RNP dynamics. In yeast, YVH1 has been shown to be involved in the biogenesis of the 60S ribosome subunit and regulate cell growth. Furthermore, we have determined that its expression regulates cell cycle progression in human cell lines. Human YVH1 is widely expressed in human tissues and its gene has been found amplified in a wide variety of late stage malignancies. Our results showing that hYVH1 can function as a regulator of cellular proliferation and as a cell survival factor coupled with its role in ribosome biogenesis, tempts us to speculate that hYVH1 is an important regulator of ribotoxic cellular events.
Roberto J, Sykes CE, Vacratsis PO. (2020) Characterization of Phosphopeptide Positional Isomers on the Transcriptional Co-activator TAZ. Biochemistry. 2020 Oct 21. doi: 10.1021/acs.biochem.0c00521. Epub ahead of print.
Eshraghi M#, Gombar R#, De Repentigny Y, Vacratsis PO*, Kothary R*. (2019) Pathologic Alterations in the Proteome of Synaptosomes from a Mouse Model of Spinal Muscular Atrophy. J Proteome Res. 18, 3042-3051.
Steevensz A, Gombar R, Vergilino R, Cristescu ME, Vacratsis PO. (2018) Proteomic Profile of Daphnia pulex using Data-Independent Acquisition Mass Spectrometry and Ion Mobility Separation. Proteomics. 18, (16):e1700460.
Geng Q, Xhabija B, Knuckle C, Bonham CA, Vacratsis PO. (2017) The Atypical Dual Specificity Phosphatase hYVH1 Associates with Multiple Ribonucleoprotein Particles. J Biol Chem. 292, 539-550
Gombar R, Pitcher TE, Lewis JA, Auld J, Vacratsis PO. (2017) Proteomic characterization of seminal plasma from alternative reproductive tactics of Chinook salmon (Oncorhynchus tswatchysha). J Proteomics. 157, 1-9.
Xhabija B, Vacratsis PO. (2015) Receptor-mediated Endocytosis 8 Utilizes an N-terminal Phosphoinositide-binding Motif to Regulate Endosomal Clathrin Dynamics. J Biol Chem. 290, 21676-89
Bonham CA, Steevensz AJ, Geng Q, Vacratsis PO. (2014) Investigating redox regulation of protein tyrosine phosphatases using low pH thiol labeling and enrichment strategies coupled to MALDI-TOF mass spectrometry. Methods. 65, 190-200
Franklin NE, Bonham CA, Xhabija B, Vacratsis PO. (2013) Differential phosphorylation of the phosphoinositide 3-phosphatase MTMR2 regulates its association with early endosomal subtypes. J Cell Sci. 126, 1333-44
Franklin NE, Taylor GS, Vacratsis PO. (2011) Endosomal Targeting of the Phosphoinositide 3-Phosphatase MTMR2 is Regulated by an N-Terminal Phosphorylation site. J Biol Chem, 286, 15841-53
Kozarova A, Hudson JW, Vacratsis PO. (2011) The Dual Specificity Phosphatase hYVH1 (DUSP12) is a Novel Modular of Cellular DNA Content. Cell Cycle. 10, 1669-78
Xhabija B, Taylor GS, Fujibayashi A, Sekiguchi K, Vacratsis PO. (2011) Receptor mediated endocytosis 8 is a novel PI(3)P Binding Protein Regulated by MTMR2. FEBS Letters. 585, 1722-8.
Faccenda A, Bonham CA, Vacratsis PO, Zhang X, Mutus B. (2010) Gold Nanoparticle Enrichment Method for Identifying S-Nitrosylation and S-Glutathionylation Sites in Proteins. J Am Chem Soc., 132, 11392-4
Bonham CA, Vacratsis PO. (2009) Redox regulation of the dual specificity phosphatase hYVH1 through disulfide bond formation. J Biol Chem., 284, 22853-64
Sharda PR, Bonham CA, Mucaki EJ, Butt Z, Vacratsis PO. (2009) The dual-specificity phosphatase hYVH1 interacts with Hsp70 and prevents heat-shock-induced cell death. Biochem J. 418, 391-401.