Structural Basis for the Catalytic Activity of Human Serine/Threonine Protein Phosphatase-5

Swingle, Mark R.; Honkanen, Richard E.; Ciszak, Ewa

doi:10.1074/jbc.m402855200

Cited by 103 publications

(161 citation statements)

References 68 publications

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“…A new water molecule fills the cavity left by one of the missing oxygen atoms of the phosphate. The conformations of almost all PP5 residues coordinating Cdc37-S13E, the active site water molecules, and the Mn 2+ ions are unchanged compared with the phosphate-bound holo structure (17). The exception is Arg275, for which the guanadinium group has flipped.…”

Section: Resultsmentioning

confidence: 81%

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Structural and functional basis of protein phosphatase 5 substrate specificity

Oberoi

Dunn

Woodford

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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The serine/threonine phosphatase protein phosphatase 5 (PP5) regulates hormone-and stress-induced cellular signaling by association with the molecular chaperone heat shock protein 90 (Hsp90). PP5-mediated dephosphorylation of the cochaperone Cdc37 is essential for activation of Hsp90-dependent kinases. However, the details of this mechanism remain unknown. We determined the crystal structure of a Cdc37 phosphomimetic peptide bound to the catalytic domain of PP5. The structure reveals PP5 utilization of conserved elements of phosphoprotein phosphatase (PPP) structure to bind substrate and provides a template for many PPP-substrate interactions. Our data show that, despite a highly conserved structure, elements of substrate specificity are determined within the phosphatase catalytic domain itself. Structure-based mutations in vivo reveal that PP5-mediated dephosphorylation is required for kinase and steroid hormone receptor release from the chaperone complex. Finally, our data show that hyper-or hypoactivity of PP5 mutants increases Hsp90 binding to its inhibitor, suggesting a mechanism to enhance the efficacy of Hsp90 inhibitors by regulation of PP5 activity in tumors.Hsp90 | PP5 | Cdc37 | chaperone | phosphatase P rotein phosphatase 5 (PP5) has pleiotropic roles in cellular signaling, including DNA damage repair, proliferation of breast cancer cells, circadian cycling, response to cytotoxic stresses, Rac-dependent potassium ion channel activity, and activation of steroid hormone receptors [e.g., glucocorticoid receptor (GR) and estrogen receptor] (1, 2). It is a member of the phosphoprotein phosphatase (PPP) family of serine/threonine phosphatases, which has members that share a highly conserved catalytic core and catalytic mechanism dependent on two metal ions, commonly Mn 2+ . Most PPP family members exhibit high, nonspecific phosphatase activity. Specificity is provided by a large cohort of regulatory and other interacting proteins that function to inhibit basal activity and recruit substrates, thereby finely tuning the enzymes (3). This combinatorial approach enables a small number of catalytic subunits to have the breadth of specificity equivalent to that seen in kinases, which are greater in number by an order of magnitude. Structures of complexes between regulatory and catalytic domains have illuminated the importance of regulatory subunits in facilitating substrate recruitment (3). However, to date, there is no structural information describing how a substrate binds at the active site of a PPP; therefore, a central question remains of how local interactions between the substrate and the catalytic domain contribute to the molecular basis of dephosphorylation.PP5 is unique among the PPP family because it has a low basal activity caused by an autoinhibitory N-terminal tetratricopeptide (TPR) domain (4). Its activity is promoted by a number of cellular factors, including fatty acids and the molecular chaperone heat shock protein 90 (Hsp90) (5), both of which release autoinhibition by interacting with the TPR...

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Section: Resultsmentioning

confidence: 81%

“…The exception is Arg275, for which the guanadinium group has flipped. The conformation of the Cdc37 peptide is, therefore, that of a trapped substrate, because all residues and active site waters are in orientations that are suitable for the inline nucleophilic attack proposed for the catalytic mechanism (17).…”

Section: Resultsmentioning

confidence: 99%

Structural and functional basis of protein phosphatase 5 substrate specificity

Oberoi

Dunn

Woodford

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…Subsequent studies revealed that cantharidin functions as an inhibitor of several PPP family serine/threonine protein phosphatases (PPases), acting as a weak inhibitor of calcineurin (PP2B) and a potent inhibitor of three families (PP1, PP2A, and PP5) of structurally related PPases (14 -16). Recent structural studies revealed that the cantharidin-sensitive PPases share a common catalytic mechanism (17), and structure/activity relationship studies indicate that cantharidin (15) and fostriecin (18), another natural product with antitumor activity, both act to inhibit the catalytic activity of the same PPP family phosphatases (15,17,18). Human genetic studies have identified four highly homologous isoforms of PP1 (PP1a, PP1h/y, PP1g1, and PP1g2), two nearly identical isoforms of PP2A (PP2a and PP2Ah), and two PPases (designated PP4 and PP6) that, at the level of their primary amino acid sequence, share f54% and 60% identity with PP2Aa, respectively (for review, see ref.…”

Section: Introductionmentioning

confidence: 99%

Cantharidin-induced mitotic arrest is associated with the formation of aberrant mitotic spindles and lagging chromosomes resulting, in part, from the suppression of PP2Aα

Bonness

Aragon

Rutland³

et al. 2006

Molecular Cancer Therapeutics

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Cantharidin, a natural vesicant, inhibits the activity of several PPP family phosphatases, displays antitumor activity, and induces apoptosis in many types of tumor cells. However, the molecular mechanisms underlying the antitumor activity of cantharidin are not clear. Here, doseresponse studies confirm a strong correlation between the suppression of phosphatase activity and cell death. Flow cytometry analysis indicates that before apoptosis, cantharidin delays cell cycle progression following DNA replication with no apparent effect on G 1 -S or S-G 2 phase progression. In contrast, studies with double thymidinesynchronized populations of cells indicate that cantharidin can rapidly arrest growth when added during G 2 or early M phase. Immunostaining indicates that cell cycle arrest occurs before the completion of mitosis and is associated with the appearance of aberrant mitotic spindles. Live cell imaging with time-lapse microscopy shows that cantharidin disrupts the metaphase alignment of chromosomes and produces a prolonged mitotic arrest, with the onset of apoptosis occurring before the onset of anaphase. To explore the contribution of individual phosphatases, antisense oligonucleotides and small interfering RNA were developed to suppress the expression of cantharidinsensitive phosphatases. The suppression of PP2AA, but not PP2AB, is sufficient to induce metaphase arrest, during which time lagging chromosomes are observed moving between the spindle poles and the metaphase plate. Immunostaining revealed slightly abnormal, yet predominately bipolar, mitotic spindles. Nonetheless, after a 10-to 15-hour delay, the cells enter anaphase, suggesting that an additional cantharidin-sensitive phosphatase is involved in the progression from metaphase into anaphase or to prevent the onset of apoptosis in cells arrested during mitosis.

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“…PP5 is an okadaic acid/cantharidin/microcystin-sensitive member of the PPP family of Ser/Thr phosphatases, which also includes PP1, PP2A, PP4, and PP6 (7)(8)(9). Currently the roles played by PP5 in biology and disease are not clear.…”

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confidence: 99%

Disruption of Serine/Threonine Protein Phosphatase 5 (PP5:PPP5c) in Mice Reveals a Novel Role for PP5 in the Regulation of Ultraviolet Light-induced Phosphorylation of Serine/Threonine Protein Kinase Chk1 (CHEK1)

Amable

Grankvist

Largen

et al. 2011

Journal of Biological Chemistry

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Background:The roles of PP5 in normal biology are poorly understood. Results: To help evaluate the biological actions of PP5, a Cre/loxP-conditional mouse line was generated. Conclusion: In response to UV light, PP5 regulates the phosphorylation of Chk1 at Ser-345. Significance: Understanding the biological roles for phosphatases is critical for understanding the role of reversible phosphorylation in the control of signaling networks.

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Structural Basis for the Catalytic Activity of Human Serine/Threonine Protein Phosphatase-5

Cited by 103 publications

References 68 publications

Structural and functional basis of protein phosphatase 5 substrate specificity

Structural and functional basis of protein phosphatase 5 substrate specificity

Cantharidin-induced mitotic arrest is associated with the formation of aberrant mitotic spindles and lagging chromosomes resulting, in part, from the suppression of PP2Aα

Disruption of Serine/Threonine Protein Phosphatase 5 (PP5:PPP5c) in Mice Reveals a Novel Role for PP5 in the Regulation of Ultraviolet Light-induced Phosphorylation of Serine/Threonine Protein Kinase Chk1 (CHEK1)

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