Phosphorylation/dephosphorylation of PTP-PEST at Serine 39 is crucial for cell migration.

We investigated the molecular details of the role of protein tyrosine phosphatase (PTP)-PEST in cell migration. PTP-PEST knockout mouse embryonic fibroblasts (KO MEFs) and MEF cells expressing a dominant-negative mutant of PTP-PEST showed significant suppression of cell migration compared to MEF cells expressing wild-type PTP-PEST (WT MEFs). Moreover, MEF cells harbouring a constitutively active mutant of PTP-PEST (S39A MEFs) showed a marked decrease in cell migration. In addition, MEF cells with no PTP-PEST or little PTP activity rapidly adhered to fibronectin and made many focal adhesions compared to WT MEF cells. In contrast, S39A MEF cells showed weak adhesion to fibronectin and formed a few focal adhesions. Furthermore, investigating the subcellular localization showed that Ser39-phosphorylated PTP-PEST was favourably situated in the adherent area of the pseudopodia. Therefore, we propose that suppression of PTP-PEST enzyme activity due to Ser39-phosphorylation in pseudopodia and at the leading edge of migrating cells induces rapid and good adherence to the extracellular matrix. Thus, suppression of PTP activity by Ser39-phosphorylation is critical for cell migration. Three amino acid substitutions in human PTP-PEST have been previously reported to alter PTP activity. These amino acid substitutions in mouse PTP-PEST altered the migration of MEF cells in a positive correlation.

Real-World Outcomes in Diabetic Macular Edema for the 0.2 µg/Day Fluocinolone Acetonide Implant: Case Series from the Midlands, UK.

AIM: To investigate real-world effectiveness and safety of fluocinolone acetonide (FAc) implant over three years of treatment in eyes with diabetic macular edema (DME) in a population with large ethnic diversity. METHODS: This audit of three large treatment centres in the UK involved retrospective collection of outcome data (best recorded visual acuity [BRVA] by Early Treatment Diabetic Retinopathy Study [ETDRS] letters, central retinal thickness [CRT], intraocular pressure [IOP] and use of supplementary treatments) from patients with DME treated with 0.2 µg/day FAc intravitreal implant with three-year follow-up expected. RESULTS: A total of 96 eyes were included. Ninety (93.8%) eyes had received prior intravitreal treatment. Increases in mean BRVA were significant at one, two and three years (p<0.05). Overall, 78.1% of eyes gained or maintained BRVA; just over 50% gained ≥5 letters, representing a functional response. Eleven (11.6%) patients lost ≥10 letters by year three. Decreases in central retinal thickness (CRT) nearing 200 µm in the first year were sustained to three years (p < 0.0001). Patients with baseline VA ≥60 letters maintained their BRVA throughout follow-up, while significant improvements at month 12 (p<0.0001) in those with baseline BRVA <60 letters were maintained through month 36 (p < 0.005). Fifty-three (55.2%) eyes required no supplementary therapy during follow-up. Increases in IOP to ≥30 mmHg and ≥25 mmHg were seen in 12 (12.5%) and 23 (24.0%) eyes, respectively. CONCLUSION: This study confirms the effectiveness and tolerability of FAc implant up to 36 months in a real-world setting, highlighting the importance of early treatment for sustaining functional vision for patients.

Neurologic music therapy in multidisciplinary acute stroke rehabilitation: Could it be feasible and helpful?

Background: There is increasing evidence for music-based interventions in neurorehabilitation, improving mood and functional outcomes. In response, there is growing interest from health-care providers in setting up Neurologic Music Therapy (NMT) services. This paper presents some preliminary data on the feasibility and acceptability of NMT in the acute stroke, multidisciplinary team setting, about which little is known. Objectives: To assess the feasibility and acceptability of a two-day per-week NMT service over 24 months. Methods: Data were collected on the number of referrals received, sessions attended, sessions declined and reasons why. Staff completed questionnaires, and collected them from patients and their relatives, rating interventions: 1. Not helpful, 2. Quite helpful, 3. Helpful, 4. Very helpful. Patients completed the Visual Analogue Mood Scale (VAMS) pre-/post- a single session. Results: Of 201 patients referred, 177 received treatment and 675 sessions were delivered. Twenty-four patients were discharged before sessions were scheduled and 28 sessions were declined, predominantly due to fatigue. Mean scores (SD) from questionnaire data were: patients (n = 99) 3.34 (0.825), relatives (n = 13) 3.83 (0.372), staff (n = 27) 3.85 (0.388). Mean, post-session VAMS data (n = 52) showed a non-significant reduction in 'Sad' (7.5, p = .007, CI = 2.1, 12.9) and an increase in 'Happy' (+ 6.2, p = .013, CI = -11.0, -1.4). Conclusions: Data suggest the service was feasible and helpful, particularly for patient mood, possibly improving engagement in rehabilitation. Research to determine generalizability in different stroke environments and treatment effects within them is warranted.

United Kingdom Diabetic Retinopathy Electronic Medical Record (UK DR EMR) Users Group: report 4, real-world data on the impact of deprivation on the presentation of diabetic eye disease at hospital services.

AIM: To assess the impact of deprivation on diabetic retinopathy presentation and related treatment interventions, as observed within the UK hospital eye service. METHODS: This is a multicentre, national diabetic retinopathy database study with anonymised data extraction across 22 centres from an electronic medical record system. The following were the inclusion criteria: all patients with diabetes and a recorded, structured diabetic retinopathy grade. The minimum data set included, for baseline, age and Index of Multiple Deprivation, based on residential postcode; and for all time points, visual acuity, ETDRS grading of retinopathy and maculopathy, and interventions (laser, intravitreal therapies and surgery). The main  outcome measures were (1) visual acuity and binocular visual state, and (2) presence of sight-threatening complications and need for early treatment. RESULTS: 79 775 patients met the inclusion criteria. Deprivation was associated with later presentation in patients with diabetic eye disease: the OR of being sight-impaired at entry into the hospital eye service (defined as 6/18 to better than 3/60 in the better seeing eye) was 1.29 (95% CI 1.20 to 1.39) for the most deprived decile vs 0.77 (95% CI 0.70 to 0.86) for the least deprived decile; the OR for being severely sight-impaired (3/60 or worse in the better seeing eye) was 1.17 (95% CI 0.90 to 1.55) for the most deprived decile vs 0.88 (95% CI 0.61 to 1.27) for the least deprived decile (reference=fifth decile in all cases). There is also variation in sight-threatening complications at presentation and treatment undertaken: the least deprived deciles had lower chance of having a tractional retinal detachment (OR=0.48 and 0.58 for deciles 9 and 10, 95% CI 0.24 to 0.90 and 0.29 to 1.09, respectively); in terms of accessing treatment, the rate of having a vitrectomy was lowest in the most deprived cohort (OR=0.34, 95% CI 0.19 to 0.58). CONCLUSIONS: This large real-world study suggests that first presentation at a hospital eye clinic with visual loss or sight-threatening diabetic eye disease is associated with deprivation. These initial hospital visits represent the first opportunities to receive treatment and to formally engage with support services. Such patients are more likely to be sight-impaired or severely sight-impaired at presentation, and may need additional resources to engage with the hospital eye services over complex treatment schedules.

Treating Diabetic Macular Oedema (DMO): real world UK clinical outcomes for the 0.19mg Fluocinolone Acetonide intravitreal implant (Iluvien™) at 2 years.

BACKGROUND: To compare visual function and structural improvements in pseudophakic eyes with diabetic macular oedema (DMO) treated with the 0.19mg Fluocinolone Acetonide (FAc) intravitreal implant (IluvienTM) in a 'real world' setting. METHODS: A single centre retrospective evaluation of patients with DMO unresponsive to conventional treatment treated with the FAc implant according to UK guidelines. Primary efficacy endpoint was best corrected visual acuity (BCVA); secondary endpoints included optical coherence tomography evaluations of the macula (a) central retinal and (b) peak macular thickness collected at annual time points. Primary safety endpoint was new rise in IOP >27mmHg or glaucoma surgery. Patients with <1 year follow-up were excluded. RESULTS: Twenty-nine eyes were included, with mean(SD) follow up of 792(270) days. Improvement in BCVA and reduction in macular oedema was noted at all timepoints. Mean improvement in BCVA from baseline was 6 ETDRS letters at year 1(n=29), 6.5L at year 2(n=22) and 11L at year 3(n=6). Mean central retinal thickness at baseline was 451 microns, 337 microns at year 1, 342 microns at year 2 and 314 microns at year 3. Two eyes required IOP-lowering drops post implant. Supplementary treatment for persistence or recurrence of DMO was necessary in 18 eyes over the total study period of 3 years with mean time to supplementary treatment being 12 months. CONCLUSIONS: Our evaluation of the 0.19mg FAc implant delivered in a real-world setting, provides additional evidence that it is effective and safe in the treatment of patients with DMO, and can provide sustained benefit for patients with previously refractory disease.

Exercise Intensity-Dependent Effects on Cognitive Control Function during and after Acute Treadmill Running in Young Healthy Adults.

The idea that physical activity differentially impacts upon performance of various cognitive tasks has recently gained increased interest. However, our current knowledge about how cognition is altered by acute physical activity is incomplete. To measure how different intensity levels of physical activity affect cognition during and after 1 bout of physical activity, 30 healthy, young participants were randomized to perform a not-X continuous performance test (CPT) during low (LI)- and moderate intensity (MI) running. The same participants were subsequently randomized to perform the not-X CPT post LI, MI, and high intensity (HI) running. In addition, exercise related mood changes were assessed through a self-report measure pre and post running at LI, MI, and HI. Results showed worsening of performance accuracy on the not-X CPT during one bout of moderate compared to low intensity running. Post running, there was a linear decrease in reaction time with increasing running intensity and no change in accuracy or mood. The decreased reaction times post HI running recovered back to baseline within 20 min. We conclude that accuracy is acutely deteriorated during the most straining physical activity while a transient intensity-dependent enhancement of cognitive control function is present following physical activity.

Neoplastic leptomeningeal disease masquerading as central serous retinopathy. A case report.

A 69-year-old man became aware of people's speech being out of synch with their lip movements alongside persistent headaches, both of which progressively worsened. A few weeks later, he developed progressive and painless visual loss in one eye. Initial neurological evaluation, inflammatory markers and head computed tomography scan were normal. Ophthalmological examination and OCT scan revealed right macular subretinal fluid with choroidal indentation, which prompted urgent further investigations including head MRI revealing extensive leptomeningeal disease. The patient continued to deteriorate and deceased shortly afterwards. This is the first reported case of neoplastic leptomeningeal disease presenting with loss of vision due to choroidal metastasis with localised exudative retinal detachment. Diagnosing neoplastic leptomeningeal disease requires a high index of suspicion from the treating physician. Symptoms may be nonspecific and/or subtle. Combining cerebrospinal fluid cytology from lumbar puncture with contrast-enhanced magnetic resonance imaging of the brain is considered the optimal diagnostic approach.

Social deprivation as a risk factor for late presentation of proliferative diabetic retinopathy.

PURPOSE: The aim of this study was to determine whether social deprivation is a risk factor for late presentation of patients with proliferative diabetic retinopathy and whether it affects their access to urgent laser treatment. METHODS: Using a 2:1 case: control design, 102 patients referred to a UK teaching hospital as part of the UK Diabetic Retinopathy National Screening Programme were identified for the period between 1 June 2010 to 1 June 2013. Social deprivation was scored using the Index of Multiple Deprivation 2010. Additional variables considered included age, duration of disease, ethnicity, and HbA1c at time of referral. RESULTS: The cases comprised 34 patients referred with proliferative (grade R3) retinopathy with a control group of 68 patients with lower retinopathy grades; two control patients were excluded due to incomplete data. On univariate analysis, R3 retinopathy was associated with higher social deprivation (P<0.001, Mann-Whitney U-test), and with higher HbA1c (11.5% vs 8.4%; P<0.001, Mann-Whitney U-test). Forward stepwise multivariable analysis showed that the association of R3 retinopathy with deprivation was significant even after adjusting for HbA1c (P=0.016). On univariate analysis South Asian ethnicity was also identified as being a risk factor for presentation with R3 retinopathy, but this was no longer significant when HbA1c was adjusted for in a forward stepwise logistic regression analysis. CONCLUSION: In our cohort social deprivation appears to be associated with late presentation of proliferative diabetic retinopathy. Our study supports the need to target these groups to reduce preventable blindness and to identify strategies which overcome barriers to care.

Lowering the limit: reducing the CD4 T-cell threshold for ophthalmic screening in patients with HIV in an ethnically diverse UK population.

BACKGROUND: Before highly active antiretroviral therapy, cytomegalovirus (CMV) retinitis was a major threat to vision in individuals with HIV. We investigate whether ophthalmic screening of asymptomatic HIV patients still has value in the highly active antiretroviral therapy era and consider CD4 thresholds in line with the world literature and UK experience. METHODS: A retrospective chart review was conducted of all patients seen by the HIV Ophthalmic Service of a UK university hospital both before (2007-2008) and after (2011-2012) introduction of a threshold of CD4 lower than 100 cells/mm(3). Data collected included CMV and HIV RNA load, CD4 cell counts and CD4 percentage, CMV-immunoglobulin G status, ocular symptoms, and evidence of HIV-related ocular disease. RESULTS: In total, 54 patients were referred to the HIV ophthalmic service. Three patients failed to attend, resulting in complete data for 51 patients (n=24 for 2007-2008; n=27 for 2011-2012). Seven patients had ophthalmic manifestations of their HIV; these cases had lower CD4 counts than those with normal examinations (median [interquartile range], 9 [7-80] versus 175 [44-394]; P=0.0039; Mann-Whitney test). Six cases had HIV retinopathy without sight loss; one case had sight-threatening CMV retinitis associated with a CD4 count of 6 cells/mm(3). CONCLUSION: Before 2008, our practice was to screen all asymptomatic patients with CD4 counts lower than 200 cells/mm(3). Screening asymptomatic patients with CD4 counts below 100 cells/mm(3) was not associated with any missed or late-presenting cases of CMV retinitis in our HIV population.

The role of social deprivation in severe neovascular age-related macular degeneration.

BACKGROUND/AIMS: Advances in therapy have improved outcomes for patients with neovascular age-related macular degeneration (nAMD). Prompt access to treatment is a priority and may be used as a key performance indicator. In this study, we investigate how social deprivation may impact on access to services, treatment and visual impairment registration. METHODS: Patients were identified retrospectively through the Certificate of Visual Impairment system for the University Hospitals Birmingham Medical Retina service. The Index of Multiple Deprivation (IMD) 2007 score was calculated for each patient. The impact of deprivation, age, gender and ethnicity on key stages in the care pathway was assessed. RESULTS: 120 patients were identified. Patients with greater social deprivation were under-represented, had worse visual acuity at first presentation (correlation of the better-seeing eye with IMD 0.225 (p=0.013)) and had sight-impairment registration earlier (correlation -0.246; p=0.007). Deprivation did not affect time to first appointment, and was not associated with a higher rate of non-attendance. CONCLUSIONS: The late presentation and under-representation of patients with greater social deprivation is a serious concern. Our study strongly suggests that this vulnerable group is encountering barriers in accessing treatment in nAMD, and that these occur prior to entry into the Hospital Eye Service.

Protein tyrosine phosphatase-PEST (PTP-PEST) regulates mast cell-activating signals in PTP activity-dependent and -independent manners.

Aggregation of the high-affinity IgE receptor (FcεRI) in mast cells leads to degranulation and production of numerous cytokines and lipid mediators that promote allergic inflammation. Tyrosine phosphorylation of proteins in response to FcεRI aggregation has been implicated in mast cell activation. Here, we determined the role of PTP-PEST (encoded by PTPN12) in the regulation of mast cell activation using the RBL-2H3 rat basophilic leukemia cell line as a model. PTP-PEST expression was significantly induced upon FcεRI-crosslinking, and aggregation of FcεRI induced the phosphorylation of PTP-PEST at Ser39, thus resulting in the suppression of PTP activity. By overexpressing a phosphatase-dead mutant (PTP-PEST CS) and a constitutively active mutant (PTP-PEST SA) in RBL-2H3 cells, we showed that PTP-PEST decreased degranulation and enhanced IL-4 and IL-13 transcription in FcεRI-crosslinked RBL-2H3 cells, but PTP activity of PTP-PEST was not necessary for this regulation. However, FcεRI-induced TNF-α transcription was increased by the overexpression of PTP-PEST SA and suppressed by the overexpression of PTP-PEST CS. Taken together, these results suggest that PTP-PEST is involved in the regulation of FcεRI-mediated mast cell activation through at least two different processes represented by PTP activity-dependent and -independent pathways.

A global pharmaceutical company initiative: an evidence-based approach to define the upper limit of body weight loss in short term toxicity studies.

Short term toxicity studies are conducted in animals to provide information on major adverse effects typically at the maximum tolerated dose (MTD). Such studies are important from a scientific and ethical perspective as they are used to make decisions on progression of potential candidate drugs, and to set dose levels for subsequent regulatory studies. The MTD is usually determined by parameters such as clinical signs, reductions in body weight and food consumption. However, these assessments are often subjective and there are no published criteria to guide the selection of an appropriate MTD. Even where an objective measurement exists, such as body weight loss (BWL), there is no agreement on what level constitutes an MTD. A global initiative including 15 companies, led by the National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs), has shared data on BWL in toxicity studies to assess the impact on the animal and the study outcome. Information on 151 studies has been used to develop an alert/warning system for BWL in short term toxicity studies. The data analysis supports BWL limits for short term dosing (up to 7days) of 10% for rat and dog and 6% for non-human primates (NHPs).

Immunomodulatory role of proteinase-activated receptor-2.

OBJECTIVE: Proteinase-activated receptor-2 (PAR(2)) has been implicated in inflammatory articular pathology. Using the collagen-induced arthritis model (CIA) the authors have explored the capacity of PAR(2) to regulate adaptive immune pathways that could promote autoimmune mediated articular damage. METHODS: Using PAR(2) gene deletion and other approaches to inhibit or prevent PAR(2) activation, the development and progression of CIA were assessed via clinical and histological scores together with ex vivo immune analyses. RESULTS: The progression of CIA, assessed by arthritic score and histological assessment of joint damage, was significantly (p<0.0001) abrogated in PAR(2) deficient mice or in wild-type mice administered either a PAR(2) antagonist (ENMD-1068) or a PAR(2) neutralising antibody (SAM11). Lymph node derived cell suspensions from PAR(2) deficient mice were found to produce significantly less interleukin (IL)-17 and IFNγ in ex vivo recall collagen stimulation assays compared with wild-type littermates. In addition, substantial inhibition of TNFα, IL-6, IL-1ß and IL-12 along with GM-CSF and MIP-1α was observed. However, spleen and lymph node histology did not differ between groups nor was any difference detected in draining lymph node cell subsets. Anticollagen antibody titres were significantly lower in PAR(2) deficient mice. CONCLUSION: These data support an important role for PAR(2) in the pathogenesis of CIA and suggest an immunomodulatory role for this receptor in an adaptive model of inflammatory arthritis. PAR(2) antagonism may offer future potential for the management of inflammatory arthritides in which a proteinase rich environment prevails.

Opportunities to minimise animal use in pharmaceutical regulatory general toxicology: a cross-company review.

Toxicity studies in animals are carried out to identify the intrinsic hazard of a substance to support risk assessment for humans. In order to identify opportunities to minimise animal use in regulatory toxicology studies, a review of current study designs was carried out. Pharmaceutical companies and contract research organisations in the UK shared data and experience of standard toxicology studies (ranging from one to nine months duration) in rodents and non-rodents; and carcinogenicity studies in the rat and mouse. The data show that variation in study designs was primarily due to (i) the number of animals used in the main study groups, (ii) the use of animals in toxicokinetic (TK) satellite groups, and (iii) the use of animals in off-treatment recovery groups. The information has been used to propose a series of experimental designs where small adjustments could reduce animal use in practice, while maintaining the scientific objectives.

Inhibition of the Unfolded Protein Response by metformin in renal proximal tubular epithelial cells.

Metformin (Met), an AMP-activated protein kinase (AMPK) inducer, is primarily transported by organic cation transporters expressed at the surface of renal proximal tubular epithelial cells. However, the implication of Met in renal function remains poorly understood. Interestingly, AICAR, another AMPK inducer, has been shown to inhibit the Unfolded Protein Response (UPR) generated by tunicamycin in cardiomyocytes in an AMPK-kinase dependent fashion suggesting metformin may also block the UPR. In this work, we have examined the effect of metformin on the expression of UPR-related markers (GRP94 and CHOP) induced by glucosamine (GlcN), 2-deoxyglucose (2-DOG) and tunicamycin (TUNI) in renal proximal tubular epithelial cells and in murine mesangial cells. Met attenuated GRP94 and CHOP expression induced by GlcN and 2-DOG, but not TUNI only in renal epithelial cells, even though the AMPK activation was observed in both renal epithelial and mesangial cells. Met did not require the contribution of its AMPK kinase inducing activity to block UPR markers expression. This report has identified a novel inhibitory function of metformin on UPR, which may have a beneficial impact on kidney homeostatic function.

Global expression study in colorectal cancer on proteins with alkaline isoelectric point by two-dimensional difference gel electrophoresis.

Colorectal cancer is one of the leading causes of cancer death worldwide. To identify candidates for biomarkers and therapeutic targets, we investigated the proteome of colorectal cancer tissues. Using 2D-DIGE in combination with our original large format electrophoresis apparatus, we compared surgically resected normal and tumor tissues from 53 patients with colorectal cancer. We focused on proteins with an alkaline pI using IPG gels for the alkaline range. We observed 1687 protein spots, and found 100 spots with statistical (p<0.01) and significant (>2-fold) differences between the normal and the tumor tissue groups. Among these 100 protein spots, five showed a different intensity between tumor tissues from the stage-II and the stage-III patients. MS experiments revealed that these 100 protein spots corresponded to 58 unique proteins. These included six proteins which had not been previously reported to be associated with colorectal cancer. Among these proteins, five were not reported in any type of malignancy. IEF/western blotting confirmed the differences in protein expression between the normal and the tumor tissues. These results may provide an insight for biomarker development and drug target discovery in colorectal cancer.

Optogenetic fMRI sheds light on the neural basis of the BOLD signal.

Blood oxygenation level dependent (BOLD) functional magnetic resonance imaging (fMRI) is widely used as a measure of neuronal activity, despite an incomplete understanding of the hemodynamic and neural bases for BOLD signals. Recent work by Lee and colleagues investigated whether activating genetically specified neurons elicits BOLD responses. Integrating optogenetic control of specific cells and fMRI showed that stimulating excitatory neurons triggers a positive BOLD signal with conventional kinetics locally and delayed weaker BOLD signals distally.

Reduced fractional anisotropy does not change the shape of the hemodynamic response in survivors of severe traumatic brain injury.

The hemodynamic response (HDR) function is the basis for standard functional magnetic resonance imaging (fMRI) analysis. HDR is influenced by white matter inflammation. Traumatic brain injury (TBI) is frequently accompanied by diffuse white matter injury, but the effect of this on the HDR has not been investigated. The aims of the present study were to describe the HDR in visual cortex and examine its relationship with the microstructure of the optic radiation in severe TBI survivors and controls. Ten severe TBI survivors without visual impairments, but with known diffuse axonal injury, and 9 matched controls underwent diffusion tensor imaging (DTI) and fMRI. From the fMRI time series obtained during brief randomized visual stimuli, blood oxygenation level-dependent (BOLD) signal changes for each subject were estimated in V1, and group HDR curves were produced. Standard between-group analysis of BOLD activation in V1 + V2 was performed. For each individual the optic radiations were identified and fractional anisotropy (FA) plus mean apparent diffusion coefficient (ADC(mean)) values for these tracts were calculated. Group HDR curves from the visual cortex were fully transposable between TBI survivors and controls, despite a significant reduction in FA in the optic radiation in TBI survivors. A significant correlation between BOLD signal in the visual cortex and FA values in the optical tract was present in controls, but not in TBI survivors. Between-group comparisons showed that TBI survivors had increased areas of activation in V1 and V2. The HDR appears to be intact in traumatic white matter damage, supporting the validity of using standard fMRI methodology to study neuroplasticity in TBI.

Protein phosphatase 1alpha associates with protein tyrosine phosphatase-PEST inducing dephosphorylation of phospho-serine 39.

Protein tyrosine phosphatase (PTP)-PEST is expressed in a wide variety of several cell types and is an efficient regulator of cell adhesion, spreading and migration. PTP-PEST-associating molecules are important in elucidating the function of PTP-PEST. Herein, we have identified protein phosphatase 1alpha (PP1alpha) as a novel PTP-PEST binding protein, and then we aimed to determine how PP1alpha contributes to the phosphorylation at Ser39 of PTP-PEST, whose phosphorylation suppresses PTP-PEST enzymatic activity. The HEK 293 cells overexpressing exogenous PTP-PEST were stimulated by 12-O-tetradecanoylphorbol 13-acetate (TPA) and the phosphorylation of PTP-PEST at Ser39 was evaluated using an anti-phospho-Ser39 PTP-PEST specific antibody (anti-pS39-PEST Ab). It was demonstrated that the phosphorylation at Ser39 detected by anti-pS39-PEST Ab was dependent on TPA treatment and a significant inverse correlation between the PTP activity of PTP-PEST and anti-pS39-PEST Ab-immunoreactive band intensity. The phosphorylation of Ser39 was suppressed by co-transfection of a plasmid encoding wild-type PP1alpha, but not by that of the dominant-negative PP1alpha mutant. Furthermore, TPA-induced phosphorylation could take place in PTP-PEST catalytic domain, but the phosphorylation of PTP-PEST catalytic domain could not be abrogated by co-transfection of a plasmid expressing wild-type PP1alpha. In conclusion, PP1alpha associates with the non-catalytic domain of PTP-PEST and regulates PTP activity via dephosphorylation of phospho-Ser39.