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1.  Protein phosphatase 5 protects neurons against amyloid β toxicity 
Journal of neurochemistry  2009;111(2):391-402.
Amyloid β (Aβ) is thought to promote neuronal cell loss in Alzheimer’s disease (AD), in part through the generation of reactive oxygen species (ROS) and subsequent activation of mitogen-activated protein kinase (MAPK) pathways. Protein phosphatase 5 (PP5) is a ubiquitously expressed serine/threonine phosphatase which has been implicated in several cell stress response pathways and shown to inactivate MAPK pathways through key dephosphorylation events. Therefore we examined whether PP5 protects dissociated embryonic rat cortical neurons in vitro from cell death evoked by Aβ. As predicted, neurons in which PP5 expression was decreased by siRNA treatment were more susceptible to Aβ toxicity. In contrast, overexpression of PP5, but not the inactive PP5 mutant, H304Q, prevented MAPK phosphorylation and neurotoxicity induced by Aβ. PP5 also prevented cell death caused by direct treatment with H2O2, but did not prevent Aβ-induced production of ROS. Thus, the neuroprotective effect of PP5 requires its phosphatase activity and lies downstream of Aβ-induced generation of ROS. In summary, our data indicate that PP5 plays a pivotal neuroprotective role against cell death induced by Aβ and oxidative stress. Consequently, PP5 might be an effective therapeutic target in AD and other neurodegenerative disorders in which oxidative stress is implicated.
doi:10.1111/j.1471-4159.2009.06337.x
PMCID: PMC3044491  PMID: 19686245
PP5; protein phosphatase 5; amyloid β; Alzheimer’s disease; neuroprotection; oxidative stress
2.  Novel Ser/Thr Protein Phosphatase 5 (PP5) Regulated Targets during DNA Damage Identified by Proteomics Analysis 
The DNA damage response is a global phosphorylation signaling cascade process involved in sensing the damaged DNA condition and coordinating responses to cope with and repair the perturbed cellular state. We utilized a label-free liquid chromatography-mass spectrometry approach to evaluate changes in protein phosphorylation associated with PP5 activity during the DNA damage response. Biological replicate analyses of bleomycin-treated HeLa cells expressing either WT-PP5 or mutant inactive PP5 lead to the identification of six potential target proteins of PP5 action. Four of these putative targets are known to be involved in DNA damage responses. Using phospho-site specific antibodies, we confirmed that phosphorylation of one target, ribosomal protein S6, was selectively decreased in cells overexpressing catalytically inactive PP5. Our findings also suggest that PP5 may play a role in controlling translation and in regulating substrates for proline-directed kinases, such as MAP kinases and cyclin-dependent protein kinases that are involved in response to DNA damage.
doi:10.1021/pr9008207
PMCID: PMC2818885  PMID: 20039704
Label-free quantitation; DNA damage; Comparative phosphoproteomics; Immobilized metal ion affinity chromatography (IMAC); Mass spectrometry (MS); nano reverse phase HPLC; Protein phosphatase 5; PP5; Ser/Thr protein phosphatase; Bleomycin
3.  The Influence of Sample Preparation and Replicate Analyses on HeLa Cell Phosphoproteome Coverage 
Journal of proteome research  2008;7(6):2215-2221.
Ongoing optimization of proteomic methodologies seeks to improve both the coverage and confidence of protein identifications. The optimization of sample preparation, inclusion of technical replicates (repeated instrumental analysis of the same sample), and biological replicates (multiple individual samples) is crucial in proteomic studies to avoid the pitfalls associated with single point analysis and under-sampling. Phosphopeptides were isolated from HeLa cells and analyzed by nano-reversed phase liquid chromatography electrospray ionization tandem mass spectrometry (nano-RP-LC-MS/MS). We observed that a detergent-based protein extraction approach, followed with additional steps for nucleic acid removal, provided a simple alternative to the broadly used Trizol extraction. The evaluation of four technical replicates demonstrated measurement reproducibility with low percent variance in peptide responses at approximately 3%, where additional peptide identifications were made with each added technical replicate. The inclusion of six technical replicates for moderately complex protein extracts (approximately 4000 uniquely identified peptides per dataset) affords the optimal collection of peptide information.
doi:10.1021/pr700575m
PMCID: PMC2517255  PMID: 18412383
Comparative phosphoproteomics; Immobilized metal-ion affinity chromatography (IMAC); Mass spectrometry; 50 μm ID reversed phase column; 1D SDS-PAGE; Non-metal nano-HPLC
4.  Applying a Targeted Label-free Approach using LC-MS AMT Tags to Evaluate Changes in Protein Phosphorylation Following Phosphatase Inhibition 
Journal of proteome research  2007;6(11):4489-4497.
In order to identify phosphoproteins regulated by the phosphoprotein phosphatase (PPP) family of S/T phosphatases, we performed a large-scale characterization of changes in protein phosphorylation on extracts from HeLa cells treated with or without calyculin A, a potent PPP enzyme inhibitor. A label-free comparative phosphoproteomics approach using immobilized metal ion affinity chromatography and targeted tandem mass spectrometry was employed to discover and identify signatures based upon distinctive changes in abundance. Overall, 232 proteins were identified as either direct or indirect targets for PPP enzyme regulation. Most of the present identifications represent novel PPP enzyme targets at the level of both phosphorylation site and protein. These data can be used to define the underlying signaling pathways and events regulated by the PPP family of S/T phosphatases.
doi:10.1021/pr070068e
PMCID: PMC2516346  PMID: 17929957
Label-free quantitation; Targeted MS/MS; AMT tag pipeline; Comparative phosphoproteomics; Immobilized metal ion affinity chromatography (IMAC); Mass spectrometry; 20 μm ID monolithic column; Phosphoprotein phosphatase (PPP) family; Ser/Thr protein phosphatase; Calyculin A
5.  Calcium and Vitamin D increase mRNA levels for the growth control hIK1 channel in human epidermal keratinocytes but functional channels are not observed 
BMC Dermatology  2004;4:7.
Background
Intermediate-conductance, calcium-activated potassium channels (IKs) modulate proliferation and differentiation in mesodermal cells by enhancing calcium influx, and they contribute to the physiology of fluid movement in certain epithelia. Previous reports suggest that IK channels stimulate proliferative growth in a keratinocyte cell line; however, because these channels indirectly promote calcium influx, a critically unique component of the keratinocyte differentiation program, an alternative hypothesis is that they would be anti-proliferative and pro-differentiating. This study addresses these hypotheses.
Methods
Real-time PCR, patch clamp electrophysiology, and proliferation assays were used to determine if human IK1 (hIK1) expression and function are correlated with either proliferation or differentiation in cultured human skin epidermal keratinocytes, and skin biopsies grown in explant culture.
Results
hIK1 mRNA expression in human keratinocytes and skin was increased in response to anti-proliferative/pro-differentiating stimuli (elevated calcium and Vitamin D). Correspondingly, the hIK1 agonist 1-EBIO inhibited keratinocyte proliferation suggesting that the channel could be anti-proliferative and pro-differentiating. However, this proliferative inhibition by 1-EBIO was not reversed by a panel of hIK1 blockers, calling into question the mechanism of 1-EBIO action. Subsequent patch clamp electrophysiological analysis failed to detect hIK1 channel currents in keratinocytes, even those expressing substantial hIK1 mRNA in response to calcium and Vitamin D induced differentiation. Identical electrophysiological recording conditions were then used to observe robust IK1 currents in fibroblasts which express IK1 mRNA levels comparable to those of keratinocytes. Thus, the absence of observable hIK1 currents in keratinocytes was not a function of the electrophysiological techniques.
Conclusion
Human keratinocyte differentiation is stimulated by calcium mobilization and influx, and differentiation stimuli coordinately upregulate mRNA levels of the calcium-activated hIK1 channel. This upregulation is paradoxical in that functional hIK1 channels are not observed in cultured keratinocytes. It appears, therefore, that hIK1 does not contribute to the functional electrophysiology of primary human keratinocytes, nor intact human skin. Further, the results indicate caution is required when interpreting experiments utilizing pharmacological hIK1 modulators in human keratinocytes.
doi:10.1186/1471-5945-4-7
PMCID: PMC446203  PMID: 15200683
6.  Characterization of Saccharomyces cerevisiae protein Ser/Thr phosphatase T1 and comparison to its mammalian homolog PP5 
BMC Cell Biology  2003;4:3.
Background
Protein Ser/Thr phosphatase 5 (PP5) and its Saccharomyces cerevisiae homolog protein phosphatase T1 (Ppt1p) each contain an N-terminal domain consisting of several tetratricopeptide repeats (TPRs) and a C-terminal catalytic domain that is related to the catalytic subunits of protein phosphatases 1 and 2A, and calcineurin. Analysis of yeast Ppt1p could provide important clues to the function of PP5 and its homologs, however it has not yet been characterized at the biochemical or cellular level.
Results
The specific activity of recombinant Ppt1p toward the artificial substrates 32P-myelin basic protein (MBP) and 32P-casein was similar to that of PP5. Dephosphorylation of 32P-MBP, but not 32P-casein, was stimulated by unsaturated fatty acids and by arachidoyl coenzyme A. Limited proteolysis of Ppt1p removed the TPR domain and abrogated lipid stimulation. The remaining catalytic fragment exhibited a two-fold increase in activity toward 32P-MBP, but not 32P-casein. Removal of the C terminus increased Ppt1p activity toward both substrates two fold, but did not prevent further stimulation of activity toward 32P-MBP by lipid treatment. Ppt1p was localized throughout the cell including the nucleus. Levels of PPT1 mRNA and protein peaked in early log phase growth.
Conclusions
Many characteristics of Ppt1p are similar to those of PP5, including stimulation of phosphatase activity with some substrates by lipids, and peak expression during periods of rapid cell growth. Unlike PP5, however, proteolytic removal of the TPR domain or C-terminal truncation only modestly increased its activity. In addition, C-terminal truncation did not prevent further activation by lipid. This suggests that these regions play only a minor role in controlling its activity compared to PP5. Ppt1p is present in both the nucleus and cytoplasm, indicating that it may function in multiple compartments. The observation that Ppt1p is most highly expressed during early log phase growth suggests that this enzyme is involved in cell growth or its expression is controlled by metabolic or nutritional signals.
doi:10.1186/1471-2121-4-3
PMCID: PMC153538  PMID: 12694636

Results 1-6 (6)