Publications by authors named "Sandra Rossie"

18 Publications

  • Page 1 of 1

Tale of the Good and the Bad Cdk5: Remodeling of the Actin Cytoskeleton in the Brain.

Mol Neurobiol 2018 Apr 13;55(4):3426-3438. Epub 2017 May 13.

Department of Biochemistry, Purdue University, West Lafayette, IN, 47907, USA.

Cdk5 kinase, a cyclin-dependent kinase family member, is a key regulator of cytoskeletal remodeling in the brain. Cdk5 is essential for brain development during embryogenesis. After birth, it is essential for numerous neuronal processes such as learning and memory formation, drug addiction, pain signaling, and long-term behavior changes, all of which rely on rapid alterations in the cytoskeleton. Cdk5 activity is deregulated in various brain disorders including Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and ischemic stroke, resulting in profound remodeling of the neuronal cytoskeleton, loss of synapses, and ultimately neurodegeneration. This review focuses on the "good and bad" Cdk5 in the brain and its pleiotropic contribution in regulating neuronal actin cytoskeletal remodeling. A vast majority of physiological and pathological Cdk5 substrates are associated with the actin cytoskeleton. Thus, our special emphasis is on the numerous Cdk5 substrates identified in the past two decades such as ephexin1, p27, Mst3, CaMKv, kalirin-7, RasGRF2, Pak1, WAVE1, neurabin-1, TrkB, 5-HT6R, talin, drebrin, synapsin I, synapsin III, CRMP1, GKAP, SPAR, PSD-95, and LRRK2. These substrates have unraveled the molecular mechanisms by which Cdk5 plays divergent roles in regulating neuronal actin cytoskeletal dynamics both in healthy and diseased states.
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http://dx.doi.org/10.1007/s12035-017-0525-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6370010PMC
April 2018

Dnmt3b Methylates DNA by a Noncooperative Mechanism, and Its Activity Is Unaffected by Manipulations at the Predicted Dimer Interface.

Biochemistry 2018 07 4;57(29):4312-4324. Epub 2016 Nov 4.

Department of Biochemistry, Purdue University Center for Cancer Research , Purdue University , West Lafayette , Indiana 47907 , United States.

The catalytic domains of the de novo DNA methyltransferases Dnmt3a-C and Dnmt3b-C are highly homologous. However, their unique biochemical properties could potentially contribute to differences in the substrate preferences or biological functions of these enzymes. Dnmt3a-C forms tetramers through interactions at the dimer interface, which also promote multimerization on DNA and cooperativity. Similar to the case for processive enzymes, cooperativity allows Dnmt3a-C to methylate multiple sites on the same DNA molecule; however, it is unclear whether Dnmt3b-C methylates DNA by a cooperative or processive mechanism. The importance of the tetramer structure and cooperative mechanism is emphasized by the observation that the R882H mutation in the dimer interface of DNMT3A is highly prevalent in acute myeloid leukemia and leads to a substantial loss of its activity. Under conditions that distinguish between cooperativity and processivity, we show that in contrast to that of Dnmt3a-C, the activity of Dnmt3b-C is not cooperative and confirm the processivity of Dnmt3b-C and the full length Dnmt3b enzyme. Whereas the R878H mutation (mouse homologue of R882H) led to the loss of cooperativity of Dnmt3a-C, the activity and processivity of the analogous Dnmt3b-C R829H variant were comparable to those of the wild-type enzyme. Additionally, buffer acidification that attenuates the dimer interface interactions of Dnmt3a-C had no effect on Dnmt3b-C activity. Taken together, these results demonstrate an important mechanistic difference between Dnmt3b and Dnmt3a and suggest that interactions at the dimer interface may play a limited role in regulating Dnmt3b-C activity. These new insights have potential implications for the distinct biological roles of Dnmt3a and Dnmt3b.
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http://dx.doi.org/10.1021/acs.biochem.6b00964DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5992102PMC
July 2018

Cdc14 phosphatases preferentially dephosphorylate a subset of cyclin-dependent kinase (Cdk) sites containing phosphoserine.

J Biol Chem 2012 Jan 23;287(3):1662-9. Epub 2011 Nov 23.

Department of Biochemistry, Purdue Center for Cancer Research, Purdue University, West Lafayette, Indiana 47907, USA.

Mitotic cell division is controlled by cyclin-dependent kinases (Cdks), which phosphorylate hundreds of protein substrates responsible for executing the division program. Cdk inactivation and reversal of Cdk-catalyzed phosphorylation are universal requirements for completing and exiting mitosis and resetting the cell cycle machinery. Mechanisms that define the timing and order of Cdk substrate dephosphorylation remain poorly understood. Cdc14 phosphatases have been implicated in Cdk inactivation and are thought to be generally specific for Cdk-type phosphorylation sites. We show that budding yeast Cdc14 possesses a strong and unusual preference for phosphoserine over phosphothreonine at Pro-directed sites in vitro. Using serine to threonine substitutions in the Cdk consensus sites of the Cdc14 substrate Acm1, we demonstrate that phosphoserine specificity exists in vivo. Furthermore, it appears to be a conserved property of all Cdc14 family phosphatases. An invariant active site residue was identified that sterically restricts phosphothreonine binding and is largely responsible for phosphoserine selectivity. Optimal Cdc14 substrates also possessed a basic residue at the +3 position relative to the phosphoserine, whereas substrates lacking this basic residue were not effectively hydrolyzed. The intrinsic selectivity of Cdc14 may help establish the order of Cdk substrate dephosphorylation during mitotic exit and contribute to roles in other cellular processes.
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http://dx.doi.org/10.1074/jbc.M111.281105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3265848PMC
January 2012

Novel Ser/Thr protein phosphatase 5 (PP5) regulated targets during DNA damage identified by proteomics analysis.

J Proteome Res 2010 Feb;9(2):945-53

Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington 99352, USA.

The DNA damage response likely includes a global phosphorylation signaling cascade process for 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 have been previously reported 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.
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http://dx.doi.org/10.1021/pr9008207DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2818885PMC
February 2010

Activated Rac1 GTPase translocates protein phosphatase 5 to the cell membrane and stimulates phosphatase activity in vitro.

J Biol Chem 2010 Feb 30;285(6):3872-3882. Epub 2009 Nov 30.

From the Department of Biochemistry and Center for Cancer Research, Purdue University, West Lafayette, Indiana 47907 and. Electronic address:

Physiological studies of ion channel regulation have implicated the Ser/Thr protein phosphatase 5 (PP5) as an effector of Rac1 GTPase signaling, but direct biochemical evidence for PP5 regulation by Rac1 is lacking. In this study we used immunoprecipitation, in vitro binding, cellular fractionation, and immunofluorescence techniques to show that the tetratricopeptide repeat domain of PP5 interacts specifically and directly with active Rac1. Consequently, activation of Rac1 promoted PP5 translocation to the plasma membrane in intact cells and stimulated PP5 phosphatase activity in vitro. In contrast, neither constitutively active RhoA-V14 nor dominant negative Rac1N17, which preferentially binds GDP and retains an inactive conformation, bound PP5 or stimulated its activity. In addition, Rac1N17 and Rac1(PBRM), a mutant lacking the C-terminal polybasic region required for Rac1 association with the membrane, both failed to cause membrane translocation of PP5. Mutation of predicted contact residues in the PP5 tetratricopeptide repeat domain or within Rac1 also disrupted co-immunoprecipitation of Rac1-PP5 complexes and membrane translocation of PP5. Specific binding of PP5 to activated Rac1 provides a direct mechanism by which PP5 can be stimulated and recruited to participate in Rac1-mediated signaling pathways.
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http://dx.doi.org/10.1074/jbc.M109.088427DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2823530PMC
February 2010

Protein phosphatase 5 protects neurons against amyloid-beta toxicity.

J Neurochem 2009 Oct 17;111(2):391-402. Epub 2009 Aug 17.

Department of Biochemistry and Purdue Cancer Center, Purdue University, West Lafayette, Indiana 47907, USA.

Amyloid-beta (Abeta) is thought to promote neuronal cell loss in Alzheimer's disease, 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 Abeta. As predicted, neurons in which PP5 expression was decreased by small-interfering RNA treatment were more susceptible to Abeta toxicity. In contrast, over-expression of PP5, but not the inactive mutant, PP5(H304Q), prevented MAPK phosphorylation and neurotoxicity induced by Abeta. PP5 also prevented cell death caused by direct treatment with H(2)O(2), but did not prevent Abeta-induced production of ROS. Thus, the neuroprotective effect of PP5 requires its phosphatase activity and lies downstream of Abeta-induced generation of ROS. In summary, our data indicate that PP5 plays a pivotal neuroprotective role against cell death induced by Abeta and oxidative stress. Consequently, PP5 might be an effective therapeutic target in Alzheimer's disease and other neurodegenerative disorders in which oxidative stress is implicated.
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http://dx.doi.org/10.1111/j.1471-4159.2009.06337.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3044491PMC
October 2009

The influence of sample preparation and replicate analyses on HeLa Cell phosphoproteome coverage.

J Proteome Res 2008 Jun 16;7(6):2215-21. Epub 2008 Apr 16.

Biological Sciences Division and Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, Washington 99352, USA.

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) are 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 data set) affords the optimal collection of peptide information.
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http://dx.doi.org/10.1021/pr700575mDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2517255PMC
June 2008

Applying a targeted label-free approach using LC-MS AMT tags to evaluate changes in protein phosphorylation following phosphatase inhibition.

J Proteome Res 2007 Nov 12;6(11):4489-97. Epub 2007 Oct 12.

Biological Sciences Division, Pacific Northwest National Laboratory, P.O. Box 999, Richland, Washington 99352, USA.

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 include phosphorylation sites within signaling proteins such as p120 Catenin, A Kinase Anchoring Protein 8, JunB, and Type II Phosphatidyl Inositol 4 Kinase. These data can be used to define underlying signaling pathways and events regulated by the PPP family of S/T phosphatases.
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http://dx.doi.org/10.1021/pr070068eDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2516346PMC
November 2007

Cellular co-localization of protein phosphatase 5 and glucocorticoid receptors in rat brain.

Brain Res 2006 Sep 8;1111(1):1-11. Epub 2006 Aug 8.

Department of Biochemistry, Purdue University, West Lafayette, IN 47907, USA.

Glucocorticoid receptors are widely expressed in brain, where they are thought to play a role in controlling neurogenesis and to mediate many of the central nervous system effects of stress. In non-neuronal cells, protein phosphatase 5 (PP5) has been found in complexes with heat shock protein 90 and glucocorticoid receptors and may be a negative modulator of glucocorticoid receptor function. In the present study, we used co-immunofluorescence analysis to examine whether PP5 and glucocorticoid receptors are co-expressed at the cellular level in rat brain. In several regions containing major populations of glucocorticoid receptor expressing neurons, PP5 and glucocorticoid receptors were co-localized at the cellular level. These include pyramidal cells of the hippocampal CA1 and CA2 regions and dentate gyrus granule cells, cerebellar Purkinje neurons, cortical pyramidal neurons, neurons of the central nucleus of the amygdala and parvocellular neurons of the hypothalamic paraventricular nucleus. There are also neuronal populations that are stained strongly for glucocorticoid receptors, such as cerebellar granule cells, where PP5 is either absent or below detection limits. Likewise, numerous neuronal populations contain PP5, but not glucocorticoid receptors. Whereas glucocorticoid receptors are expressed in both neurons and glial cells throughout the brain, PP5 appears to be primarily expressed in neurons. These studies suggest that glucocorticoid receptors may be differentially regulated by phosphatase action in different populations of central nervous system cells. Co-localization of PP5 and glucocorticoid receptors in brain regions involved in feedback control of the hypothalamus-pituitary-adrenal axis suggests that PP5 may be an important modulator of glucocorticoid receptor responses in this pathway.
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http://dx.doi.org/10.1016/j.brainres.2006.06.106DOI Listing
September 2006

More sensitive and quantitative proteomic measurements using very low flow rate porous silica monolithic LC columns with electrospray ionization-mass spectrometry.

J Proteome Res 2006 May;5(5):1091-7

Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington 99352, USA.

The sensitivity of proteomics measurements using liquid chromatography (LC) separations interfaced with electrospray ionization-mass spectrometry (ESI-MS) improves approximately inversely with liquid flow rate (for the columns having the same separation efficiency, linear velocity, and porosity), making attractive the use of smaller inner diameter LC columns. We report the development and initial application of 10 microm i.d. silica-based monolithic LC columns providing more sensitive proteomics measurements. A 50-microm-i.d. micro solid-phase extraction precolumn was used for ease of sample injection and cleanup prior to the reversed-phase LC separation, enabling the sample volume loading speed to be increased by approximately 50-fold. Greater than 10-fold improvement in sensitivity was obtained compared to analyses using more conventional capillary LC, enabling e.g. the identification of >5000 different peptides by MS/MS from 100-ng of a Shewanella oneidensis tryptic digest using an ion trap MS. The low nL/min LC flow rates provide more uniform responses for different peptides, and provided improved quantitative measurements compared to conventional separation systems without the use of internal standards or isotopic labeling. The improved sensitivity allowed LC-MS measurements of immunopurified protein phosphatase 5 that were in good agreement with quantitative Western blot analyses.
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http://dx.doi.org/10.1021/pr050424yDOI Listing
May 2006

Inhibition of PP2A, but not PP5, mediates p53 activation by low levels of okadaic acid in rat liver epithelial cells.

J Cell Biochem 2006 Sep;99(1):241-55

Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907, USA.

The microbial toxin okadaic acid (OA) specifically inhibits PPP-type ser/thr protein phosphatases. OA is an established tumor promoter with numerous cellular effects that include p53-mediated cell cycle arrest. In T51B rat liver epithelial cells, a model useful for tumor promotion studies, p53 activation is induced by tumor-promoting (low nanomolar) concentrations of OA. Two phosphatases sensitive to these concentrations of OA, PP2A and protein phosphatase 5 (PP5), have been implicated as negative regulators of p53. In this study we examined the respective roles of these phosphatases in p53 activation in non-neoplastic T51B cells. Increases in p53 activity were deduced from levels of p21 (cip1) and/or the rat orthologue of mdm2, two p53-regulated gene products whose induction was blocked by siRNA-mediated knockdown of p53. As observed with 10 nM OA, both phospho-ser15-p53 levels and p53 activity were increased by 10 microM fostriecin or SV40 small t-antigen. Both of these treatments selectively inhibit PP2A but not PP5. siRNA-mediated knockdown of PP2A, but not PP5, also increased p53 activity. Finally, adenoviral-mediated over-expression of an OA-resistant form of PP5 did not prevent increased phospho-ser15-p53, p53 protein, or p53 activity caused by 10 nM OA. Together these results indicate that PP5 blockade is not responsible for OA-induced p53 activation and G1 arrest in T51B cells. In contrast, specific blockade of PP2A mimics p53-related responses to OA in T51B cells, suggesting that PP2A is the target for this response to OA.
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http://dx.doi.org/10.1002/jcb.20919DOI Listing
September 2006

Rac GTPase signaling through the PP5 protein phosphatase.

Proc Natl Acad Sci U S A 2006 Mar 20;103(13):5202-6. Epub 2006 Mar 20.

Environmental Biology Program, National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, NC 27709, USA.

We have investigated the Rac-dependent mechanism of KCNH2 channel stimulation by thyroid hormone in a rat pituitary cell line, GH(4)C(1), with the patch-clamp technique. Here we present physiological evidence for the protein serine/threonine phosphatase, PP5, as an effector of Rac GTPase signaling. We also propose and test a specific molecular mechanism for PP5 stimulation by Rac-GTP. Inhibition of PP5 with the microbial toxin, okadaic acid, blocked channel stimulation by thyroid hormone and by Rac, but signaling was restored by expression of a toxin-insensitive mutant of PP5, Y451A, which we engineered. PP5 is unique among protein phosphatases in that it contains an N-terminal regulatory domain with three tetratricopeptide repeats (TPR) that inhibit its activity. Expression of the TPR domain coupled to GFP blocked channel stimulation by the thyroid hormone. We also show that the published structures of the PP5 TPR domain and the TPR domain of p67, the Rac-binding subunit of NADPH oxidase, superimpose over 92 alpha carbons. Mutation of the PP5 TPR domain at two predicted contact points with Rac-GTP prevents the TPR domain from functioning as a dominant negative and blocks the ability of Y451A to rescue signaling in the presence of okadaic acid. PP5 stimulation by Rac provides a unique molecular mechanism for the antagonism of Rho-dependent signaling through protein kinases in many cellular processes, including metastasis, immune cell chemotaxis, and neuronal development.
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http://dx.doi.org/10.1073/pnas.0600080103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1458818PMC
March 2006

Targeted biomarker detection via whole protein ion trap tandem mass spectrometry: thymosin beta4 in a human lung cancer cell line.

J Mass Spectrom 2005 Apr;40(4):444-51

Department of Chemistry, Purdue University, West Lafayette, Indiana 47907-2084, USA.

N-Terminally acetylated thymosin beta4, a species implicated for use as a cancer biomarker, was identified in a human lung cancer cell line using ion trap tandem mass spectrometry at the whole protein level. Ion-ion proton transfer reactions were used for parent ion concentration/manipulation and to simplify interpretation of product ion spectra. Dissociation data for the +6 to +3 charge states are reported. As is usually the case, structural information available from the ion trap collisional activation of the protein is sensitive to parent ion charge state. Each parent ion charge state selected, however, provided sufficient information to make a confident identification. Furthermore, each charge state provided relatively rich fragmentation. Therefore, any of the charge states can be used to detect with high specificity thymosin beta(4) in a complex protein mixture. There are advantages associated with the rapid detection of protein biomarkers at the whole protein level, as opposed to the peptide level following protein digestion, particularly for relatively small protein and polypeptide biomarkers. Having identified and characterized the protein, product ion spectra obtained directly, without recourse to ion-ion proton transfer reactions, can be used for library matching. However, ion-ion proton transfer reactions for parent ion concentration and charge state purification are advantageous in addressing relatively complex mixtures.
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http://dx.doi.org/10.1002/jms.797DOI Listing
April 2005

Dephosphorylation of tau by protein phosphatase 5: impairment in Alzheimer's disease.

J Biol Chem 2005 Jan 15;280(3):1790-6. Epub 2004 Nov 15.

New York State Institute for Basic Research in Developmental Disabilities, Staten Island, New York 10314, USA.

Protein phosphatase (PP) 5 is highly expressed in the mammalian brain, but few physiological substrates have yet been identified. Here, we investigated the kinetics of dephosphoryation of phospho-tau by PP5 and found that PP5 had a K(m) of 8-13 microm toward tau, which is similar to that of PP2A, the major known tau phosphatase. This K(m) value is within the range of intraneuronal tau concentration in human brain, suggesting that tau could be a physiological substrate of both PP5 and PP2A. PP5 dephosphorylated tau at all 12 Alzheimer's disease (AD)-associated abnormal phosphorylation sites studied, with different efficiency toward each site. Thr(205), Thr(212), and Ser(409) of tau were the most favorable sites; Ser(199), Ser(202), Ser(214), Ser(396), and Ser(404) were less favorable sites; and Ser(262) was the poorest site for PP5. Overexpression of PP5 in PC12 cells resulted in dephosphorylation of tau at multiple phosphorylation sites. The activity but not the protein level of PP5 was found to be decreased by approximately 20% in AD neocortex. These results suggest that tau is probably a physiological substrate of PP5 and that the abnormal hyperphosphorylation of tau in AD might result in part from the decreased PP5 activity in the diseased brains.
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http://dx.doi.org/10.1074/jbc.M410775200DOI Listing
January 2005

Calcium and Vitamin D increase mRNA levels for the growth control hIK1 channel in human epidermal keratinocytes but functional channels are not observed.

BMC Dermatol 2004 Jun 16;4. Epub 2004 Jun 16.

Molecular Human Physiology Group, Fujisawa Research Institute of America, Evanston, IL 60201 USA.

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.
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http://dx.doi.org/10.1186/1471-5945-4-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC446203PMC
June 2004

Dephosphorylation of microtubule-associated protein tau by protein phosphatase 5.

J Neurochem 2004 Jan;88(2):298-310

New York State Institute for Basic Research in Developmental Disabilities, Staten Island, New York 10314, USA.

Protein phosphatase 5 (PP5) is a 58-kDa novel phosphoseryl/phosphothreonyl protein phosphatase. It is ubiquitously expressed in all mammalian tissues examined, with a high level in the brain, but little is known about its physiological substrates. We found that this phosphatase dephosphorylated recombinant tau phosphorylated with cAMP-dependent protein kinase and glycogen synthase kinase-3beta, as well as abnormally hyperphosphorylated tau isolated from brains of patients with Alzheimer's disease. The specific activity of PP5 toward tau was comparable to those reported with other protein substrates examined to date. The PP5 activity toward tau was stimulated by arachidonic acid by 30- to 45-fold. Immunostaining demonstrated that PP5 was primarily cytoplasmic in PC12 cells and in neurons of postmortem human brain tissue. A small pool of PP5 associated with microtubules. Expression of active PP5 in PC12 cells resulted in reduced phosphorylation of tau, suggesting that PP5 can also dephosphorylate tau in cells. These results suggest that PP5 plays a role in the dephosphorylation of tau and might be involved in the molecular pathogenesis of Alzheimer's disease.
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http://dx.doi.org/10.1111/j.1471-4159.2004.02147.xDOI Listing
January 2004

Characterization of Saccharomyces cerevisiae protein Ser/Thr phosphatase T1 and comparison to its mammalian homolog PP5.

BMC Cell Biol 2003 Mar 28;4. Epub 2003 Mar 28.

Department of Biochemistry, Purdue University, West Lafayette, IN 47907, USA.

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.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC153538PMC
http://dx.doi.org/10.1186/1471-2121-4-3DOI Listing
March 2003

Interacting effects of N-terminal variation and strex exon splicing on slo potassium channel regulation by calcium, phosphorylation, and oxidation.

J Biol Chem 2002 Jul 16;277(30):27045-52. Epub 2002 May 16.

Laboratory of Signal Transduction, NIEHS, National Institutes of Health, 111 Alexander Drive, Research Triangle Park, NC 27709, USA.

We have investigated the structural basis for the phenotype of a native rat Slo (rSlo) potassium channel (BK(Ca); KCNMA1) in a rat pituitary cell line, GH(4)C(1). Opposing regulation of these calcium- and voltage-activated potassium channels by cAMP- and cGMP-dependent protein kinases requires an alternatively spliced exon (strex) of 59 amino acids in the cytoplasmic C terminus of the pore-forming alpha subunit encoded by rslo. However, inclusion of this cysteine-rich exon produces a 10-fold increase in the sensitivity of the channels to inhibition by oxidation. Inclusion of the strex exon also increases channel sensitivity to stimulation by calcium, but responses in the physiological ranges of calcium and voltage require coassembly with beta(1) subunits. With strex present, however, beta(1) subunits only stimulated channels assembled from rSlo alpha subunits with a truncated N terminus beginning MDALI-. Thus N-terminal variation and strex exon splicing in rSlo interact to produce BK(Ca) channels with a physiologically relevant phenotype.
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http://dx.doi.org/10.1074/jbc.M203087200DOI Listing
July 2002