Src-mediated phosphorylation of the ribosome biogenesis factor hYVH1 affects its localization, promoting partitioning to the 60S ribosomal subunit

Abstract

Yeast VH1-related phosphatase (YVH1) (also known as DUSP12) is a member of the atypical dual-specificity phosphatase subfamily. Although no direct substrate has been firmly established, human YVH1 (hYVH1) has been shown to protect cells from cellular stressors, regulate the cell cycle, disassemble stress granules, and act as a 60S ribosome biogenesis factor. Despite knowledge of hYVH1 function, further research is needed to uncover mechanisms of its regulation. In this study, we investigate cellular effects of a Src-mediated phosphorylation site at Tyr179 on hYVH1. We observed that this phosphorylation event attenuates localization of hYVH1 to stress granules, enhances shuttling of hYVH1 to the nucleus, and promotes hYVH1 partitioning to the 60S ribosomal subunit. Quantitative proteomics reveal that Src coexpression with hYVH1 reduces formation of ribosomal species that represent stalled intermediates through the alteration of associating factors that mediate translational repression. Collectively, these results implicate hYVH1 as a novel Src substrate and provide the first demonstrated role of tyrosine phosphorylation regulating the activity of a YVH1 ortholog. Moreover, the ribosome proteome alterations point to a collaborative function of hYVH1 and Src in maintaining translational fitness. Yeast VH1-related phosphatase (YVH1) (also known as DUSP12) is a member of the atypical dual-specificity phosphatase subfamily. Although no direct substrate has been firmly established, human YVH1 (hYVH1) has been shown to protect cells from cellular stressors, regulate the cell cycle, disassemble stress granules, and act as a 60S ribosome biogenesis factor. Despite knowledge of hYVH1 function, further research is needed to uncover mechanisms of its regulation. In this study, we investigate cellular effects of a Src-mediated phosphorylation site at Tyr179 on hYVH1. We observed that this phosphorylation event attenuates localization of hYVH1 to stress granules, enhances shuttling of hYVH1 to the nucleus, and promotes hYVH1 partitioning to the 60S ribosomal subunit. Quantitative proteomics reveal that Src coexpression with hYVH1 reduces formation of ribosomal species that represent stalled intermediates through the alteration of associating factors that mediate translational repression. Collectively, these results implicate hYVH1 as a novel Src substrate and provide the first demonstrated role of tyrosine phosphorylation regulating the activity of a YVH1 ortholog. Moreover, the ribosome proteome alterations point to a collaborative function of hYVH1 and Src in maintaining translational fitness. In 1992, Guan et al. (1Guan K. Hakes D.J. Wang Y. Park H.D. Cooper T.G. Dixon J.E. A yeast protein phosphatase related to the vaccinia virus VH1 phosphatase is induced by nitrogen starvation.Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 12175-12179Crossref PubMed Scopus (78) Google Scholar) cloned and characterized the yeast VH1-related phosphatase (YVH1), marking it as one of the earliest eukaryotic dual-specificity phosphatases (DUSPs) identified. The mRNA levels of YVH1 were found to be dramatically induced by nitrogen starvation and low temperatures. In addition, knockdown of the yvh1 gene in yeast exhibited a slow growth phenotype, with defects in glycogen accumulation and spore maturation (2Beeser A.E. Cooper T.G. The dual-specificity protein phosphatase Yvh1p regulates sporulation, growth, and glycogen accumulation independently of catalytic activity in Saccharomyces cerevisiae via the cyclic AMP-dependent protein kinase cascade.J. Bacteriol. 2000; 182: 3517-3528Crossref PubMed Scopus (34) Google Scholar). Following these studies in yeast, the human ortholog (hYVH1, also known as DUSP12) was identified and shown to share 31% sequence identity with YVH1 (3Muda M. Manning E.R. Orth K. Dixon J.E. Identification of the human YVH1 protein-tyrosine phosphatase orthologue reveals a novel zinc binding domain essential for in vivo function.J. Biol. Chem. 1999; 274: 23991-23995Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Interestingly, the yvh1 gene has now been shown to be widely conserved throughout evolution in eukaryotes from yeast to humans (1Guan K. Hakes D.J. Wang Y. Park H.D. Cooper T.G. Dixon J.E. A yeast protein phosphatase related to the vaccinia virus VH1 phosphatase is induced by nitrogen starvation.Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 12175-12179Crossref PubMed Scopus (78) Google Scholar, 2Beeser A.E. Cooper T.G. The dual-specificity protein phosphatase Yvh1p regulates sporulation, growth, and glycogen accumulation independently of catalytic activity in Saccharomyces cerevisiae via the cyclic AMP-dependent protein kinase cascade.J. Bacteriol. 2000; 182: 3517-3528Crossref PubMed Scopus (34) Google Scholar, 3Muda M. Manning E.R. Orth K. Dixon J.E. Identification of the human YVH1 protein-tyrosine phosphatase orthologue reveals a novel zinc binding domain essential for in vivo function.J. Biol. Chem. 1999; 274: 23991-23995Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). YVH1 orthologs are characterized by possessing an N-terminal tyrosine phosphatase domain, which possesses the characteristic CX5R motif, and a C-terminal zinc-binding domain (ZBD). These two domains are connected by a linker region of unknown function. Initial studies on hYVH1 have shown its relevance in various cellular functions, including cell survival and the cell cycle (4Sharda P.R. Bonham C.A. Mucaki E.J. Butt Z. Vacratsis P.O. The dual-specificity phosphatase hYVH1 interacts with Hsp70 and prevents heat-shock-induced cell death.Biochem. J. 2009; 418: 391-401Crossref PubMed Scopus (29) Google Scholar, 5Kozarova A. Hudson J.W. Vacratsis P.O. The dual-specificity phosphatase hYVH1 (DUSP12) is a novel modulator of cellular DNA content.Cell Cycle. 2011; 10: 1669-1678Crossref PubMed Scopus (23) Google Scholar). When investigating further into the capability of hYVH1 to protect cells from oxidative stress induced by H2O2, it was revealed that the ZBD acts as a redox sensor, capable of forming intramolecular disulfide bonds (6Bonham C.A. Vacratsis P.O. Redox regulation of the human dual specificity phosphatase YVH1 through disulfide bond formation.J. Biol. Chem. 2009; 284: 22853-22864Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). This allows for hYVH1 to avoid irreversible inactivation during severe oxidative stress by protecting the catalytic cysteine residue, again highlighting the importance of this unique ZBD. Once the stress is removed and reducing conditions have been established, zinc ejection is readily reversible (and required) to recover the intrinsic phosphatase activity of hYVH1 in vitro (6Bonham C.A. Vacratsis P.O. Redox regulation of the human dual specificity phosphatase YVH1 through disulfide bond formation.J. Biol. Chem. 2009; 284: 22853-22864Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). A central aspect of YVH1 biology has been the revelation of its role in 60S ribosome biogenesis. In yeast, YVH1 has been shown to act as a critical transacting factor responsible for exchanging the placeholder Mrt4 for the P0 stalk protein. Association of P0 is required to complete maturation of the 60S ribosomal subunit leading to efficient 80S ribosome formation. In addition to its role in 60S ribosome biogenesis, an interactome analysis recently discovered that hYVH1 associates with multiple ribonucleoprotein (RNP) complex proteins, including stress granules (7Geng Q. Xhabija B. Knuckle C. Bonham C.A. Vacratsis P.O. The atypical dual specificity phosphatase hYVH1 associates with multiple ribonucleoprotein particles.J. Biol. Chem. 2017; 292: 539-550Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). Upon further investigation, it was demonstrated that hYVH1 functions as a stress granule disassembly factor (7Geng Q. Xhabija B. Knuckle C. Bonham C.A. Vacratsis P.O. The atypical dual specificity phosphatase hYVH1 associates with multiple ribonucleoprotein particles.J. Biol. Chem. 2017; 292: 539-550Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). Although, increasing evidence points to a role for hYVH1 in RNP remodeling, less is known how hYVH1 is regulated and In this study, we that the tyrosine kinase Src is to hYVH1 its linker leading to and 60S ribosome Quantitative proteomics discovered that coexpression of hYVH1 and Src to protein alterations on the 80S ribosome with a role in translational for the first that hYVH1 is regulated by tyrosine hYVH1 interactome (7Geng Q. Xhabija B. Knuckle C. Bonham C.A. Vacratsis P.O. The atypical dual specificity phosphatase hYVH1 associates with multiple ribonucleoprotein particles.J. Biol. Chem. 2017; 292: 539-550Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). these we observed to the tyrosine kinase Although these the conditions we were in investigating Src was to tyrosine on hYVH1. cells and were to and by The results demonstrated that with tyrosine phosphorylation was We also low tyrosine phosphorylation on hYVH1 cells were with growth factor Interestingly, tyrosine phosphorylation of hYVH1 was cells were with a to Src in cells the analysis and the was on hYVH1 as in the Initial identified a at to to of hYVH1. The was also identified at this including the was a that and J. Vacratsis P.O. of on the PubMed Scopus Google Scholar). Upon analysis of the phosphorylation of Tyr179 was of the to a C-terminal of a The of the the of the phosphorylation site to We also observed tyrosine phosphorylation of hYVH1 an in vitro kinase was from and hYVH1 was as the substrate by this in vitro hYVH1 was found to on Tyr179 Moreover, a kinase Src and hYVH1 revealed that Src hYVH1 in When the sequence and of Tyr179 in a at the N-terminal of the linker region the phosphatase and B. et and function of human dual-specificity protein Biol. PubMed Scopus Google Scholar). Interestingly, this tyrosine is conserved in YVH1 orthologs including yeast, it an role We have shown that hYVH1 functions as a stress granule disassembly factor (7Geng Q. Xhabija B. Knuckle C. Bonham C.A. Vacratsis P.O. The atypical dual specificity phosphatase hYVH1 associates with multiple ribonucleoprotein particles.J. Biol. Chem. 2017; 292: 539-550Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). a of hYVH1 and we demonstrated that hYVH1 readily to stress granules in to knockdown of hYVH1 formation of stress granules, disassembly was as observed by a in stress granule during stress stress granule was hYVH1 was in cells it was of to Src-mediated tyrosine phosphorylation of hYVH1 the of hYVH1 to to stress granules were induced by of and was to stress granules E.R. is a Biol. 2017; Scopus Google Scholar, M. M. et granule is by of Biol. PubMed Google Scholar). hYVH1 to stress granules in to A and Src hYVH1 to stress granules A and that phosphorylation of Tyr179 direct hYVH1 from stress granule disassembly be by stress granule as stress granules in to stress granule Interestingly, stress granules are observed hYVH1 is with Src with A and further that tyrosine phosphorylation of hYVH1 attenuates localization to stress granule and the role of hYVH1 in stress granule of Tyr179 to represent and hYVH1 were to the Src coexpression the hYVH1 a stress granule and stress granule as that the of hYVH1 the stress granule disassembly function of hYVH1 Interestingly, hYVH1 was at to stress granules, to Src with hYVH1. Moreover, stress granules in cells hYVH1 were in the analysis was via the the localization of hYVH1 to stress granules was on cells from and were and in A of complete no and a of The discovered were as stress granule with hYVH1 and the hYVH1 of and When hYVH1 was with Src and for hYVH1 were at and of with stress granules In addition, stress granule for stress the stress granules were hYVH1 hYVH1 were of and and for hYVH1 Src and hYVH1 of and these results that Src-mediated phosphorylation of hYVH1 its to with stress granules and in The that Src-mediated phosphorylation attenuates the localization of hYVH1 to stress granules that a of Tyr179 phosphorylation is to the localization of hYVH1. is that YVH1 orthologs are capable of shuttling the and S. M. is required for a maturation in the 60S biogenesis Biol. 2009; PubMed Scopus Google Scholar, Z. Wang stalk the dual-specificity phosphatase for the of Mrt4 with Biol. 2009; PubMed Scopus Google Scholar). is that this shuttling is of the role of YVH1 in 60S ribosome biogenesis, it is that YVH1 associates with the 60S subunit in the and from the 60S subunit in the S. M. is required for a maturation in the 60S biogenesis Biol. 2009; PubMed Scopus Google Scholar, Z. Wang stalk the dual-specificity phosphatase for the of Mrt4 with Biol. 2009; PubMed Scopus Google Scholar). protein phosphorylation is a regulating localization of a protein K. a PubMed Scopus Google and to the that Tyr179 phosphorylation reduces hYVH1 stress granule we were in Src-mediated phosphorylation of hYVH1 the localization of hYVH1. hYVH1 in cells a localization with in the and region coexpression with hYVH1 localization dramatically to a of the hYVH1 the Src coexpression The localization of the hYVH1 the a localization to hYVH1 with Src the on the were hYVH1 a and hYVH1 a and localization In hYVH1 with Src a and localization and hYVH1 a and localization Collectively, these results that Src-mediated phosphorylation of hYVH1 regulate the shuttling hYVH1 into the The that Src-mediated phosphorylation the localization of hYVH1 is we its role in ribosome biogenesis. The of transacting factors as hYVH1 into the has been shown to be a critical for maintaining a of ribosome biogenesis S. M. is required for a maturation in the 60S biogenesis Biol. 2009; PubMed Scopus Google Scholar, Z. Wang stalk the dual-specificity phosphatase for the of Mrt4 with Biol. 2009; PubMed Scopus Google Scholar). that Src-mediated phosphorylation results in localization that this phosphorylation event the of hYVH1 ribosome is evidence that shuttling is a of the maturation of the 60S ribosomal subunit S. M. is required for a maturation in the 60S biogenesis Biol. 2009; PubMed Scopus Google Scholar, Z. Wang stalk the dual-specificity phosphatase for the of Mrt4 with Biol. 2009; PubMed Scopus Google Scholar). YVH1 in yeast has been shown to act as a factor to Mrt4 on the subunit in the and is by the P0 the the this YVH1 is and to be into the for of 60S subunit biogenesis. to the that the of hYVH1 was in to Src it was to tyrosine phosphorylation hYVH1 with the 60S subunit. we were in ribosomal conditions that to proteome alterations induced by coexpression of hYVH1 and these ribosomal were a that of the ribosomal by through a to that was and on a at the of the by the 80S ribosomal and the The are the at is This a ribosomal that is and to of and cell a ribosomal from cells was Interestingly, a on the with the 80S was We that these 80S subunit mRNA and 80S mRNA the to of the were by the that and represent the and 60S ribosomal and The analysis for and to to the that an of and 60S ribosomal This that to the 80S also ribosomal protein identified in as as ribosomal Moreover, the of was in that the ribosomal identified was to of ribosomal in the This is with the of the ribosome and that was to and ribosomal In addition to the ribosomal subunit of the also for the of the ribosomal from to the ribosomal of the proteome that be to regulation of RNP Upon of the and of the we the ribosomal for hYVH1 with on the results Src of hYVH1 with stress granules, one is that Src-mediated phosphorylation also hYVH1 with the 60S ribosomal subunit. the Src the of hYVH1 observed in the a efficient of hYVH1 for its ribosome biogenesis function in to Tyr179 Interestingly, analysis of the by revealed that hYVH1 is with the of hYVH1 in the 60S subunit was This was in multiple that Src-mediated phosphorylation enhances of hYVH1 to the 60S ribosomal subunit. in the hYVH1 Src was the in the with hYVH1 Src and This is as a of represent stalled in translational Y. 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The localization in to Src phosphorylation of hYVH1 the of a it is also that hYVH1 is from the 60S ribosome 60S ribosomal stalk it that the proteome and that Src-mediated tyrosine phosphorylation of Tyr179 shuttling hYVH1 to the for of 60S ribosome a of be required to the of regulation of translational the have discovered proteome alterations that as for Moreover, it is to these results in the of that hYVH1 cellular as cell cell cycle and cell growth (4Sharda P.R. Bonham C.A. Mucaki E.J. Butt Z. Vacratsis P.O. The dual-specificity phosphatase hYVH1 interacts with Hsp70 and prevents heat-shock-induced cell death.Biochem. J. 2009; 418: 391-401Crossref PubMed Scopus (29) Google Scholar, 5Kozarova A. Hudson J.W. Vacratsis P.O. The dual-specificity phosphatase hYVH1 (DUSP12) is a novel modulator of cellular DNA content.Cell Cycle. 2011; 10: 1669-1678Crossref PubMed Scopus (23) Google Scholar). effects with to a that the role of hYVH1 in the of 60S ribosome biogenesis and translational the of critical for cellular The have been (3Muda M. Manning E.R. Orth K. Dixon J.E. Identification of the human YVH1 protein-tyrosine phosphatase orthologue reveals a novel zinc binding domain essential for in vivo function.J. Biol. Chem. 1999; 274: 23991-23995Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). was to hYVH1 and The and were the and for and and for and by DNA The of hYVH1 has been (4Sharda P.R. Bonham C.A. Mucaki E.J. Butt Z. Vacratsis P.O. The dual-specificity phosphatase hYVH1 interacts with Hsp70 and prevents heat-shock-induced cell death.Biochem. J. 2009; 418: 391-401Crossref PubMed Scopus (29) Google Scholar, C.A. Vacratsis P.O. Redox regulation of the human dual specificity phosphatase YVH1 through disulfide bond formation.J. Biol. Chem. 2009; 284: 22853-22864Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). was by cells were and as a in with and at and hYVH1 cells were for to with and growth factor for in the the of the cells were to into were via cells were with and in with cells were with for to cell cells were with and in and with and were from cell by at for at ribosomal cells were with for at cells were two with with cell cells were from cell by the addition of of with by cells and the cell into one were by at for at were in and in with and were in with were from cell by at for at were for ribosomal was from cellular via a on a at were in and and in for analysis by hYVH1 was from cells a to protein A from the of Dixon of of (3Muda M. Manning E.R. Orth K. Dixon J.E. Identification of the human YVH1 protein-tyrosine phosphatase orthologue reveals a novel zinc binding domain essential for in vivo function.J. Biol. Chem. 1999; 274: 23991-23995Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). the in vitro kinase from cells as (4Sharda P.R. Bonham C.A. Mucaki E.J. Butt Z. Vacratsis P.O. The dual-specificity phosphatase hYVH1 interacts with Hsp70 and prevents heat-shock-induced cell death.Biochem. J. 2009; 418: 391-401Crossref PubMed Scopus (29) Google Scholar, C.A. Vacratsis P.O. Redox regulation of the human dual specificity phosphatase YVH1 through disulfide bond formation.J. Biol. Chem. 2009; 284: 22853-22864Abstract Full Text Full Text PDF PubMed Scopus (30) Google and Src were in was from cellular an to protein A and the kinase were as Bonham C.A. Xhabija B. Vacratsis P.O. phosphorylation of the regulates its with Sci. PubMed Scopus Google Scholar). 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