2021
Dempsey, D. R.; Viennet, T.; Iwase, R.; Park, E. Y.; Henriquez, S.; Chen, Z.; Jeliazkov, J. R.; Palanski, B. A.; Phan, K. L.; Coote, P.; Gray, J. J.; Eck, M. J.; Gabelli, S. B.; Arthanari, H.; Cole, P. A.
The structural basis of PTEN regulation by multi-site phosphorylation Journal Article
In: Nat. Struct. Mol. Biol., vol. 28, no. 10, pp. 858-868, 2021, ISSN: 1545-9985.
Abstract | Links | BibTeX | Tags: Animals, Ciona intestinalis, Crystallography, Fluorescence Polarization, Humans, Magnetic Resonance Spectroscopy, Molecular Docking Simulation, Phosphorylation, PTEN Phosphohydrolase, X-Ray
@article{1624365,
title = {The structural basis of PTEN regulation by multi-site phosphorylation},
author = {D. R. Dempsey and T. Viennet and R. Iwase and E. Y. Park and S. Henriquez and Z. Chen and J. R. Jeliazkov and B. A. Palanski and K. L. Phan and P. Coote and J. J. Gray and M. J. Eck and S. B. Gabelli and H. Arthanari and P. A. Cole},
doi = {10.1038/s41594-021-00668-5},
issn = {1545-9985},
year = {2021},
date = {2021-10-28},
urldate = {2021-10-28},
journal = {Nat. Struct. Mol. Biol.},
volume = {28},
number = {10},
pages = {858-868},
abstract = {Phosphatase and tensin homolog (PTEN) is a phosphatidylinositol-3,4,5-triphosphate (PIP3) phospholipid phosphatase that is commonly mutated or silenced in cancer. PTEN’s catalytic activity, cellular membrane localization and stability are orchestrated by a cluster of C-terminal phosphorylation (phospho-C-tail) events on Ser380, Thr382, Thr383 and Ser385, but the molecular details of this multi-faceted regulation have remained uncertain. Here we use a combination of protein semisynthesis, biochemical analysis, NMR, X-ray crystallography and computational simulations on human PTEN and its sea squirt homolog, VSP, to obtain a detailed picture of how the phospho-C-tail forms a belt around the C2 and phosphatase domains of PTEN. We also visualize a previously proposed dynamic N-terminal α-helix and show that it is key for PTEN catalysis but disordered upon phospho-C-tail interaction. This structural model provides a comprehensive framework for how C-tail phosphorylation can impact PTEN’s cellular functions.},
keywords = {Animals, Ciona intestinalis, Crystallography, Fluorescence Polarization, Humans, Magnetic Resonance Spectroscopy, Molecular Docking Simulation, Phosphorylation, PTEN Phosphohydrolase, X-Ray},
pubstate = {published},
tppubtype = {article}
}
Phosphatase and tensin homolog (PTEN) is a phosphatidylinositol-3,4,5-triphosphate (PIP3) phospholipid phosphatase that is commonly mutated or silenced in cancer. PTEN’s catalytic activity, cellular membrane localization and stability are orchestrated by a cluster of C-terminal phosphorylation (phospho-C-tail) events on Ser380, Thr382, Thr383 and Ser385, but the molecular details of this multi-faceted regulation have remained uncertain. Here we use a combination of protein semisynthesis, biochemical analysis, NMR, X-ray crystallography and computational simulations on human PTEN and its sea squirt homolog, VSP, to obtain a detailed picture of how the phospho-C-tail forms a belt around the C2 and phosphatase domains of PTEN. We also visualize a previously proposed dynamic N-terminal α-helix and show that it is key for PTEN catalysis but disordered upon phospho-C-tail interaction. This structural model provides a comprehensive framework for how C-tail phosphorylation can impact PTEN’s cellular functions.
2016
Henager, Samuel H; Chu, Nam; Chen, Zan; Bolduc, David; Dempsey, Daniel R; Hwang, Yousang; Wells, James; Cole, Philip A
Enzyme-catalyzed expressed protein ligation Journal Article
In: Nat Methods, vol. 13, no. 11, pp. 925-927, 2016, ISSN: 1548-7105.
Abstract | Links | BibTeX | Tags: Animals, Bacillus subtilis, Blotting, Catalytic Domain, Cells, Cultured, Cysteine, Escherichia coli, Fibroblasts, Mice, Mutagenesis, Peptide Fragments, Peptide Synthases, Phosphorylation, Post-Translational, Protein Processing, PTEN Phosphohydrolase, Recombinant Proteins, Site-Directed, Subtilisins, Western
@article{1299949,
title = {Enzyme-catalyzed expressed protein ligation},
author = {Samuel H Henager and Nam Chu and Zan Chen and David Bolduc and Daniel R Dempsey and Yousang Hwang and James Wells and Philip A Cole},
doi = {10.1038/nmeth.4004},
issn = {1548-7105},
year = {2016},
date = {2016-11-01},
journal = {Nat Methods},
volume = {13},
number = {11},
pages = {925-927},
abstract = {Expressed protein ligation is a valuable method for protein semisynthesis that involves the reaction of recombinant protein C-terminal thioesters with N-terminal cysteine (N-Cys)-containing peptides, but the requirement of a Cys residue at the ligation junction can limit the utility of this method. Here we employ subtiligase variants to efficiently ligate Cys-free peptides to protein thioesters. Using this method, we have more accurately determined the effect of C-terminal phosphorylation on the tumor suppressor protein PTEN.},
keywords = {Animals, Bacillus subtilis, Blotting, Catalytic Domain, Cells, Cultured, Cysteine, Escherichia coli, Fibroblasts, Mice, Mutagenesis, Peptide Fragments, Peptide Synthases, Phosphorylation, Post-Translational, Protein Processing, PTEN Phosphohydrolase, Recombinant Proteins, Site-Directed, Subtilisins, Western},
pubstate = {published},
tppubtype = {article}
}
Expressed protein ligation is a valuable method for protein semisynthesis that involves the reaction of recombinant protein C-terminal thioesters with N-terminal cysteine (N-Cys)-containing peptides, but the requirement of a Cys residue at the ligation junction can limit the utility of this method. Here we employ subtiligase variants to efficiently ligate Cys-free peptides to protein thioesters. Using this method, we have more accurately determined the effect of C-terminal phosphorylation on the tumor suppressor protein PTEN.
Chen, Zan; Dempsey, Daniel R; Thomas, Stefani N; Hayward, Dawn; Bolduc, David M; Cole, Philip A
Molecular Features of Phosphatase and Tensin Homolog (PTEN) Regulation by C-terminal Phosphorylation Journal Article
In: J Biol Chem, vol. 291, no. 27, pp. 14160-9, 2016, ISSN: 1083-351X.
Abstract | Links | BibTeX | Tags: Amino Acid Sequence, Humans, Phosphorylation, PTEN Phosphohydrolase
@article{1299966,
title = {Molecular Features of Phosphatase and Tensin Homolog (PTEN) Regulation by C-terminal Phosphorylation},
author = {Zan Chen and Daniel R Dempsey and Stefani N Thomas and Dawn Hayward and David M Bolduc and Philip A Cole},
doi = {10.1074/jbc.M116.728980},
issn = {1083-351X},
year = {2016},
date = {2016-07-01},
journal = {J Biol Chem},
volume = {291},
number = {27},
pages = {14160-9},
abstract = {PTEN is a tumor suppressor that functions to negatively regulate the PI3K/AKT pathway as the lipid phosphatase for phosphatidylinositol 3,4,5-triphosphate. Phosphorylation of a cluster of Ser/Thr residues (amino acids 380-385) on the C-terminal tail serves to alter the conformational state of PTEN from an open active state to a closed inhibited state, resulting in a reduction of plasma membrane localization and inhibition of enzyme activity. The relative contribution of each phosphorylation site to PTEN autoinhibition and the structural basis for the conformational closure is still unclear. To further the structural understanding of PTEN regulation by C-terminal tail phosphorylation, we used protein semisynthesis to insert stoichiometric and site-specific phospho-Ser/Thr(s) in the C-terminal tail of PTEN. Additionally, we employed photo-cross-linking to map the intramolecular PTEN interactions of the phospho-tail. Systematic evaluation of the PTEN C-tail phospho-cluster showed autoinhibition, and conformational closure was influenced by the aggregate effect of multiple phospho-sites rather than dominated by a single phosphorylation site. Moreover, photo-cross-linking suggested a direct interaction between the PTEN C-tail and a segment in the N-terminal region of the catalytic domain. Mutagenesis experiments provided additional insights into how the PTEN phospho-tail interacts with both the C2 and catalytic domains.},
keywords = {Amino Acid Sequence, Humans, Phosphorylation, PTEN Phosphohydrolase},
pubstate = {published},
tppubtype = {article}
}
PTEN is a tumor suppressor that functions to negatively regulate the PI3K/AKT pathway as the lipid phosphatase for phosphatidylinositol 3,4,5-triphosphate. Phosphorylation of a cluster of Ser/Thr residues (amino acids 380-385) on the C-terminal tail serves to alter the conformational state of PTEN from an open active state to a closed inhibited state, resulting in a reduction of plasma membrane localization and inhibition of enzyme activity. The relative contribution of each phosphorylation site to PTEN autoinhibition and the structural basis for the conformational closure is still unclear. To further the structural understanding of PTEN regulation by C-terminal tail phosphorylation, we used protein semisynthesis to insert stoichiometric and site-specific phospho-Ser/Thr(s) in the C-terminal tail of PTEN. Additionally, we employed photo-cross-linking to map the intramolecular PTEN interactions of the phospho-tail. Systematic evaluation of the PTEN C-tail phospho-cluster showed autoinhibition, and conformational closure was influenced by the aggregate effect of multiple phospho-sites rather than dominated by a single phosphorylation site. Moreover, photo-cross-linking suggested a direct interaction between the PTEN C-tail and a segment in the N-terminal region of the catalytic domain. Mutagenesis experiments provided additional insights into how the PTEN phospho-tail interacts with both the C2 and catalytic domains.
0000
Chen, Zan; Thomas, Stefani N; Bolduc, David M; Jiang, Xuejun; Zhang, Xiangbin; Wolberger, Cynthia; Cole, Philip A
Enzymatic Analysis of PTEN Ubiquitylation by WWP2 and NEDD4-1 E3 Ligases Journal Article
In: Biochemistry, vol. 55, no. 26, pp. 3658-66, 0000, ISSN: 1520-4995.
Abstract | Links | BibTeX | Tags: Chromatography, Endosomal Sorting Complexes Required for Transport, Humans, Immunoprecipitation, Liquid, Nedd4 Ubiquitin Protein Ligases, Phosphorylation, Post-Translational, Protein Processing, PTEN Phosphohydrolase, Tandem Mass Spectrometry, Ubiquitin, Ubiquitin-Protein Ligases, Ubiquitination, X-Linked Inhibitor of Apoptosis Protein
@article{1299965,
title = {Enzymatic Analysis of PTEN Ubiquitylation by WWP2 and NEDD4-1 E3 Ligases},
author = {Zan Chen and Stefani N Thomas and David M Bolduc and Xuejun Jiang and Xiangbin Zhang and Cynthia Wolberger and Philip A Cole},
doi = {10.1021/acs.biochem.6b00448},
issn = {1520-4995},
journal = {Biochemistry},
volume = {55},
number = {26},
pages = {3658-66},
abstract = {PTEN is a lipid phosphatase that converts phosphatidylinositol 3,4,5-phosphate (PIP3) to phosphatidylinositol 4,5-phosphate (PIP2) and plays a critical role in the regulation of tumor growth. PTEN is subject to regulation by a variety of post-translational modifications, including phosphorylation on a C-terminal cluster of four Ser/Thr residues (380, 382, 383, and 385) and ubiquitylation by various E3 ligases, including NEDD4-1 and WWP2. It has previously been shown that C-terminal phosphorylation of PTEN can increase its cellular half-life. Using in vitro ubiquitin transfer assays, we show that WWP2 is more active than NEDD4-1 in ubiquitylating unphosphorylated PTEN. The mapping of ubiquitylation sites in PTEN by mass spectrometry showed that both NEDD4-1 and WWP2 can target a broad range of Lys residues in PTEN, although NEDD4-1 versus WWP2 showed a stronger preference for ubiquitylating PTEN’s C2 domain. Whereas tetraphosphorylation of PTEN did not significantly affect its ubiquitylation by NEDD4-1, it inhibited PTEN ubiquitylation by WWP2. Single-turnover and pull-down experiments suggested that tetraphosphorylation of PTEN appears to weaken its interaction with WWP2. These studies reveal how the PTEN E3 ligases WWP2 and NEDD4-1 exhibit distinctive properties in Lys selectivity and sensitivity to PTEN phosphorylation. Our findings also provide a molecular mechanism for the connection between PTEN Ser/Thr phosphorylation and PTEN’s cellular stability.},
keywords = {Chromatography, Endosomal Sorting Complexes Required for Transport, Humans, Immunoprecipitation, Liquid, Nedd4 Ubiquitin Protein Ligases, Phosphorylation, Post-Translational, Protein Processing, PTEN Phosphohydrolase, Tandem Mass Spectrometry, Ubiquitin, Ubiquitin-Protein Ligases, Ubiquitination, X-Linked Inhibitor of Apoptosis Protein},
pubstate = {published},
tppubtype = {article}
}
PTEN is a lipid phosphatase that converts phosphatidylinositol 3,4,5-phosphate (PIP3) to phosphatidylinositol 4,5-phosphate (PIP2) and plays a critical role in the regulation of tumor growth. PTEN is subject to regulation by a variety of post-translational modifications, including phosphorylation on a C-terminal cluster of four Ser/Thr residues (380, 382, 383, and 385) and ubiquitylation by various E3 ligases, including NEDD4-1 and WWP2. It has previously been shown that C-terminal phosphorylation of PTEN can increase its cellular half-life. Using in vitro ubiquitin transfer assays, we show that WWP2 is more active than NEDD4-1 in ubiquitylating unphosphorylated PTEN. The mapping of ubiquitylation sites in PTEN by mass spectrometry showed that both NEDD4-1 and WWP2 can target a broad range of Lys residues in PTEN, although NEDD4-1 versus WWP2 showed a stronger preference for ubiquitylating PTEN’s C2 domain. Whereas tetraphosphorylation of PTEN did not significantly affect its ubiquitylation by NEDD4-1, it inhibited PTEN ubiquitylation by WWP2. Single-turnover and pull-down experiments suggested that tetraphosphorylation of PTEN appears to weaken its interaction with WWP2. These studies reveal how the PTEN E3 ligases WWP2 and NEDD4-1 exhibit distinctive properties in Lys selectivity and sensitivity to PTEN phosphorylation. Our findings also provide a molecular mechanism for the connection between PTEN Ser/Thr phosphorylation and PTEN’s cellular stability.