Expression Of Phosphoprotein Enriched In Astrocytes 15 Research Articles

Overexpression of phosphoprotein enriched in astrocytes15 reverses the damage induced by propofol in hippocampal neurons.

Propofol is a general anesthetic used in surgical operations. Phosphoprotein enriched in astrocytes 15(PEA15) was initially identified in astrocytes. The present study examined the role of PEA15 in the damage induced by propofol in hippocampal neurons. A model of hippocampal neuron damage was established using 50 µmol/l propofol. Cell viability, proliferation and apoptosis of hippocampal neurons were tested by Cell Counting Kit-8 and flow cytometry. Western blotting and reverse transcription-quantitative polymerase chain reaction analysis were performed to measure the expression levels of PEA15, and additional factors involved in apoptosis or in the signaling pathway downstream of PEA15. The present results suggested that propofol significantly decreased PEA15 expression levels in hippocampal neurons. Furthermore, overexpression of PEA15 significantly increased the cell viability and cell proliferation of cells treated with propofol. Additionally, PEA15 overexpression decreased apoptosis, which was promoted by propofol. Treatment with propofol significantly decreased the protein expression levels of pro-caspase-3, B-cell lymphoma-2, phosphorylated extracellular signal-regulated kinases (ERK)1/2, ribosomal S6 kinase 2 (RSK2) and phosphorylated cAMP responsive element binding protein 1 (CREB1). However, propofol upregulated active caspase-3 and Bax expression levels. Notably, PEA15 overexpression was able to reverse the effects of propofol. Collectively, overexpression of PEA15 was able to attenuate the neurotoxicity of propofol in rat hippocampal neurons by increasing proliferation and repressing apoptosis via upregulation of the ERK-CREB-RSK2 signaling pathway.

Abstract P2-06-22: PEA15-AA, an unphosphorylatable mutant of PEA15, as a novel therapeutic gene for triple-negative breast cancer

Abstract Background: Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer characterized by a high rate of metastatic recurrence and poor prognosis. Molecular mechanism underlying the metastatic behavior of TNBC has not been well elucidated, and newer approaches addressing drivers of metastasis are crucial to improving patient outcomes. PEA15 (Phosphoprotein enriched in astrocytes-15) regulates cell proliferation, apoptosis, and autophagy. In breast cancer, PEA15 expression inhibits invasion by binding to ERK and preventing its nuclear translocation. The biological function of PEA15 is tightly regulated by its phosphorylation at Ser104 and Ser116. However, the effect of PEA15 phosphorylation status on TNBC remains unknown. In this study, we tested the hypothesis that unphosphorylated PEA15 will prevent metastasis in TNBC through inhibition of the epithelial-to-mesenchymal transition (EMT). Method: We established stable cells overexpressing unphosphorylatable (PEA15-AA) and phospho-mimetic (PEA15-DD) PEA15 mutants in MDA-MB-468 cells. To dissect specific Cellular Mechanisms regulated by PEA15 phosphorylation, we performed RT-PCR immune and metastasis arrays. In vivo mouse models were used to see effects of PEA15 phosphorylation on tumor growth. Results: The clonogenic growth of PEA15-AA–expressing cells was significantly reduced by 80% compared with empty vector-transfected cells (PEA15-V). Anchorage-independent growth, an indicator of in vivo tumorigenicity, was inhibited in cells expressing PEA15-AA by 60% compared with PEA15-V. PEA15-AA upregulated the expression of E-cadherin and decreased the expression of mesenchymal markers, suggesting that PEA15-AA reverses EMT. Compared with PEA15-V, migration and invasion of cells expressing PEA15-AA were reduced by 65% and 72%, respectively. In contrast, PEA15-DD promoted migration, invasion, and expression of mesenchymal markers. To determine the in vivo effect of PEA15-AA, we injected stable PEA15 transfectants of MDA-MB-468 cells into the mammary fat pad of NOD/SCID mice. The PEA15-DD–injected group showed greater tumor volumes than PEA15-V and PEA15-AA groups, suggesting that PEA15-AA has antitumor effects both in vitro and in vivo. From the immune and metastasis arrays, we found that expression level of IL-8, which is known to induce EMT, was greatly decreased by PEA15-AA, while IL-8 was highly expressed in PEA15-DD cells. Addition of recombinant IL-8 to the cells expressing PEA15-AA partially rescued mesenchymal characteristics, increasing migration and expression of mesenchymal markers. By contrast, IL-8 knockdown in PEA15-DD–expressing cells decreased the mesenchymal phenotype. These findings indicate that IL-8 may play an important role as a mediator of phosphorylation of PEA-15 in breast cancer cell migration and invasion and suggest that PEA15-AA inhibits the expression of IL-8, thereby reversing EMT. Conclusion: Taken together, our results show that PEA15 phosphorylation serves as an important regulator, having a dual role as an oncogene or tumor suppressor. Further studies are warranted to evaluate the impact of PEA15 phosphorylation status on metastasis in vivo. These findings support the development of PEA15-AA as a potential therapeutic strategy for TNBC. Citation Format: Park J, Chauhan G, Cohen EN, Ueno NT, Battula VL, Tripathy D, Reuben JM, Bartholomeusz C. PEA15-AA, an unphosphorylatable mutant of PEA15, as a novel therapeutic gene for triple-negative breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-06-22.

Hyperglycemia aggravates decreases of PEA-15 and its two phosphorylated forms in cerebral ischemia.

Diabetes is a metabolic health disorder and an important risk factor for stroke. Phosphoprotein enriched in astrocytes 15 (PEA-15) is a multifunctional protein modulating cell proliferation, survival, apoptosis and glucose metabolism. This study investigated whether diabetes modulates the expression of PEA-15 and two phosphorylated forms (Ser 104 and Ser 116) in middle cerebral artery occlusion (MCAO)-induced brain injury. Male Sprague-Dawley rats were administrated with streptozotocin (40 mg/kg) and were underwent right middle cerebral artery occlusion (MCAO) 4 weeks after streptozotocin injection. Brain tissues were collected 24 hr after MCAO and stained using triphenyltetrazolium chloride. Western blot analysis was performed to elucidate the expression of PEA-15 and two phosphorylated forms (Ser 104 and Ser 116) in right cerebral cortex. Infarct volume during MCAO injury was severely increased in diabetic animals compared to non-diabetic animals. We identified the decrease in PEA-15 in animals that underwent MCAO using proteomic approach. PEA-15 expression during MCAO was strongly decreased in diabetic animals compared to non-diabetic animals. Western blots analysis confirmed that diabetes exacerbated the decrease in PEA-15 expression after MCAO. Moreover, decrease in expression of phospho-PEA-15 (Ser 104 and Ser 116) was greater in diabetic than in non-diabetic animals. These results suggested that a diabetic condition may aggravate brain damage through decreasing expression of PEA-15 and phospho-PEA-15 (Ser 104 and Ser 116) in ischemic brain injury.

Abstract 2982: Antineoplastic activity of the novel CDK2/9 inhibitor CCT68127 occurs via induced anaphase catastrophe and inhibition of PEA15 phosphorylation in lung cancer

Abstract We reported the first generation CDK2/9/7 inhibitor seliciclib (R-roscovitine, CYC202) exerts antineoplastic effects against lung cancer by inducing anaphase catastrophe. In anaphase catastrophe, cells with aneuploidy (a hallmark of cancer) cannot cluster supernumerary centrosomes, triggering abnormal anaphase and apoptosis. This study now finds the new CDK2/9 inhibitor CCT68127 (Cyclacel) is a more potent and selective CDK2 inhibitor than seliciclib. CCT68127 exerted substantial antineoplastic effects against diverse lung cancer cells. It inhibited growth, caused cell cycle arrest, and induced apoptosis more than did seliciclib. Using a high throughput screen platform of 75 (57 KRAS wild-type and 18 KRAS mutant) lung cancer cell lines, those with KRAS mutation were significantly (p<0.05) more sensitive to CCT68127 than were KRAS wild-type cells. Anaphase catastrophe was triggered by CCT68127 treatment. Expression of 180 critical growth-regulatory proteins was studied before and after 6, 24, and 48 hours of CCT68127 or vehicle treatments in murine (LKR13; KRAS mutant and ED1; KRAS wild-type) and human (Hop62; KRAS mutant and H522; KRAS wild-type) lung cancer cells using a reverse phase protein array (RPPA). Regulated species included DNA repair, Hippo, and Rab GTPase proteins. The multifunctional growth regulator PEA15 (phosphoprotein enriched in astrocytes 15), exhibited marked Ser116 phosphorylation inhibition after CCT68127 treatment. This was independently confirmed by immunoblot analysis in murine and human lung cancer cells. When PEA15 was overexpressed in these cells, CCT68127 growth inhibition was antagonized, indicating its direct involvement in these effects. PEA15 knockdown using siRNAs significantly (p<0.05) repressed growth of lung cancer cells and enhanced growth inhibition after CCT68127 treatment. PEA15 immunohistochemical detection was explored in 235 human non-small lung cancers. PEA15 expression was reduced in lung cancers versus adjacent normal lung. Decreased PEA15 expression was significantly (p<0.05) associated with advanced stage and overall survival, establishing the translational importance of PEA15 expression in lung cancer. In summary, the novel CDK2/9 inhibitor, CCT68127, elicits marked antineoplastic effects in lung cancer. This occurs through mechanisms that engage anaphase catastrophe and reduce phosphorylation of the growth regulator PEA15. Citation Format: Masanori Kawakami, Xi Liu, Lisa M. Mustachio, David J. Sekula, Shanhu Hu, Lin Zheng, Jaime Rodriguez-Canales, Barbara Mino, Pamela A. Villalobos, Carmen Behrens, Ignacio I. Wistuba, Sarah J. Freemantle, Ethan Dmitrovsky. Antineoplastic activity of the novel CDK2/9 inhibitor CCT68127 occurs via induced anaphase catastrophe and inhibition of PEA15 phosphorylation in lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2982.

Abstract 648: Reduced Expression of Phosphoprotein Enriched in Astrocytes-15 (PEA-15) Increases Neointimal Hyperplasia

Neointimal hyperplasia from increased vascular smooth muscle cell (VSMC) proliferation remains a significant clinical problem. Phosphoprotein enriched in astrocytes-15 (PEA-15) is an extracellular signal-regulated kinases 1/2 (ERK1/2) binding protein. We have previously demonstrated in vitro that PEA-15 acts as a cytoplasmic “anchor” in VSMCs and prevents ERK1/2 translocation to the nucleus, decreasing proliferation. We now assess the hypothesis that expression of PEA-15 regulates the development of neointimal hyperplasia. Arterial injury was induced by wire denudation with ligation of the carotid artery in C57BL/6 mice and examined 3, 7 and 14 days after surgery. Neointima formation was significantly increased in injured arteries from day 7 compared to control arteries. PEA-15 protein expression was significantly decreased in injured arteries in comparison to control vessels 3 days after arterial injury (ratio to GAPDH - 0.29±0.10 vs. 0.91±0.1, n=3), preceding significant neointima formation at day 7. A similar decrease in PEA-15 protein and mRNA levels was observed in human saphenous vein rings incubated ex vivo in 30% serum for 14 days to induce neointimal hyperplasia (protein ratio to GAPDH - 0.36±0.05 vs. freshly isolated veins 1.34±0.20, n=5). This coincided with increases in proliferative markers. To determine a causal relationship between decreased PEA-15 expression and neointimal formation, we induced arterial injury in mice deficient in PEA-15 (PEA-15-/-). There was a significantly exaggerated neointima formation in PEA-15-/- mice 7 days after surgery compared to C57BL/6 mice at the same time point (intima-to-media ratio - 0.95±0.26 vs. 0.35±0.09, n=3-5). The mechanisms involved were investigated. Immunolocalization of ERK1/2 was assessed in primary cultured VSMCs isolated from aortae of C57BL/6 and PEA-15-/- mice. In unstimulated VSMCs from PEA-15-/- mice, there was a significant increase in nuclear staining of ERK1/2 compared with cells from C57BL/6 mice. In conclusion, decreased PEA-15 expression occurs in VSMCs during the development of neointima hyperplasia which may increase proliferation. Targeting this pathway could be beneficial in the treatment of restenosis.

Proteomic Differential Display Analysis Reveals Decreased Expression of PEA-15 and Vimentin in FABP7-Deficient Astrocytes

Fatty acid binding proteins (FABPs) are intracellular lipid chaperones which mediate the uptake, transportation and signaling roles of fatty acids. Former studies suggest that FABP7 regulates proliferation and differentiation of radial glia and astrocytes and is associated with various central nervous system diseases including glioma and neuropsychiatric diseases. However, the underlying mechanism is poorly understood. To further explore the mechanistic roles of FABP7 on astrocyte function, the proteome of astrocytes cultured from Fabp7 KO mice was compared with wild-type counterparts by two-dimensional gel electrophoresis (2-DE). We found that 16 protein spots showed differential expression intensity on 2-DE gels. Among them, seven spots appeared elevated, and nine spots appeared decreased in Fabp7 KO astrocytes. Selected spots were analyzed using liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis and their protein identity was subsequently revealed using protein databases. Four spots including phosphoprotein enriched in astrocytes 15 (PEA-15), GFAP, vimentin and FABP7 were selected for further confirmation by Western blotting. Consistently, there was a significant decrease in expression of PEA-15 and vimentin, both of which play significant roles in proliferation, differentiation, maturation, and survival of astrocytes. These results suggest the molecular mechanism how FABP7 controls astrocyte function and provide new insight to explain the role of FABP7 in glioma and reactive gliosis.

Estradiol attenuates down-regulation of PEA-15 and its two phosphorylated forms in ischemic brain injury.

Estradiol exerts a neuroprotective effect against focal cerebral ischemic injury through the inhibition of apoptotic signals. Phosphoprotein enriched in astrocytes 15 (PEA-15) is mainly expressed in brain that perform anti-apoptotic functions. This study investigated whether estradiol modulates the expression of PEA-15 and two phosphorylated forms of PEA-15 (Ser 104 and Ser 116) in middle cerebral artery occlusion (MCAO)-induced injury and glutamate exposure-induced neuronal cell death. Adult female rats were ovariectomized to remove endogenous estradiol and treated with vehicle or estradiol prior to MCAO. Focal cerebral ischemia was induced by MCAO and cerebral cortices were collected 24 h after MCAO. Western blot analysis indicated that estradiol prevents the MCAO-induced decrease in PEA-15, phospho-PEA-15 (Ser 104), phospho-PEA-15 (Ser 116). Glutamate exposure induced a reduction in PEA-15, phospho-PEA-15 (Ser 104), phospho-PEA-15 (Ser 116) in cultured neurons, whereas estradiol treatment attenuated the glutamate toxicity-induced decrease in the expression of these proteins. It has been known that phosphorylation of PEA-15 is an important step in carrying out its anti-apoptotic function. Thus, these findings suggest that the regulation of PEA-15 phosphorylation by estradiol contributes to the neuroprotective function of estradiol in ischemic brain injury.

Phosphoprotein enriched in astrocytes (PEA)-15: A potential therapeutic target in multiple disease states

Phosphoprotein enriched in astrocytes-15 (PEA-15) is a cytoplasmic protein that sits at an important junction in intracellular signalling and can regulate diverse cellular processes, such as proliferation and apoptosis, dependent upon stimulation. Regulation of these processes occurs by virtue of the unique interaction of PEA-15 with other signalling proteins. PEA-15 acts as a cytoplasmic tether for the mitogen-activated protein kinases, extracellular signal-regulated kinase 1/2 (ERK1/2) preventing nuclear localisation. In order to release ERK1/2, PEA-15 requires to be phosphorylated via several potential pathways. PEA-15 (and its phosphorylation state) therefore regulates many ERK1/2-dependent processes, including proliferation, via regulating ERK1/2 nuclear translocation. In addition, PEA-15 contains a death effector domain (DED) which allows interaction with other DED-containing proteins. PEA-15 can bind the DED-containing apoptotic adaptor molecule, Fas-associated death domain protein (FADD) which is also dependent on the phosphorylation status of PEA-15. PEA-15 binding of FADD can inhibit apoptosis as bound FADD cannot participate in the assembly of apoptotic signalling complexes. Through these protein–protein interactions, PEA-15-regulated cellular effects have now been investigated in a number of disease-related studies. Changes in PEA-15 expression and regulation have been observed in diabetes mellitus, cancer, neurological disorders and the cardiovascular system. These changes have been suggested to contribute to the pathology related to each of these disease states. As such, new therapeutic targets based around PEA-15 and its associated interactions are now being uncovered and could provide novel avenues for treatment strategies in multiple diseases.

PEA15 impairs cell migration and correlates with clinical features predicting good prognosis in neuroblastoma

ERK and RSK2 drive proliferation and invasion of many cancers. Phosphoprotein enriched in astrocytes 15 (PEA15) binds ERK and RSK2 and high PEA15 levels can impair ERK- and RSK2-dependent transcription. PEA15 expression also inversely correlates with cell motility and invasiveness. We therefore tested PEA15 effects on neuroblastoma cells in vitro. We further analyzed PEA15 expression in the context of clinical and genetic features of neuroblastoma in tumor samples to determine its correlation with disease progression. Affymetrix microarray analysis was performed using 24 different neuroblastoma cell lines. Cell lines expressing low to intermediate levels of PEA15 were chosen for in vitro functional studies. The cell line results were verified by Affymetrix analysis of three different neuroblastic tumor types (total of 110 samples) PEA15 overexpression inhibited neuroblastoma migration in vitro. We verified that inhibition of motility required PEA15 interaction with its binding partners ERK and RSK2. Additionally, synthetic inhibitors of RSK2 suppressed integrin-dependent migration. PEA15 expression correlates with clinical parameters and a 25% increase in patient survival rate. The highest PEA15 levels were found in low stage, more differentiated and less metastatic neuroblastic tumors, and correlated with lack of MYCN amplification. PEA15 blocks neuroblastoma migration through inhibition of ERK/RSK2 signaling. PEA15 expression levels correlate with favorable clinical features suggesting that PEA15 limits metastatic progression of neuroblastoma. Thus, PEA15 and its partners ERK and RSK2 are potential targets for the development of new therapeutics to impede progression of minimal residual disease in patients with high-risk neuroblastoma.

Melatonin prevents down-regulation of astrocytic phosphoprotein PEA-15 in ischemic brain injury

Melatonin functions as a free-radical scavenger and has a neuroprotective effect against ischemic brain damage. PEA-15 (phosphoprotein enriched in astrocytes 15) regulates various cellular processes including cell proliferation and apoptosis. In this study, we investigated whether melatonin regulates the levels of PEA-15 and the two phosphorylated forms of PEA-15 (Ser 104 and Ser 116) in a middle cerebral artery occlusion (MCAO)-induced injury model and neuronal cells exposed to glutamate. Adult male rats were treated with vehicle or melatonin (5 mg/kg) prior to MCAO, and cerebral cortex tissues were collected 24 h after MCAO. PEA-15 levels after ischemic brain injury were monitored using a proteomic approach. Melatonin pretreatment prevented the ischemic injury-induced reduction in PEA-15 levels. Moreover, Western blot analysis demonstrated that melatonin attenuated the ischemic injury-induced reduction in PEA-15, phospho-PEA-15 (Ser 104), and phospho-PEA-15 (Ser 116) levels. Neuronal cells exposed to glutamate showed decreased expression of PEA-15, phospho-PEA-15 (Ser 104), and phospho-PEA-15 (Ser 116), while melatonin pretreatment prevented the glutamate toxicity-induced decreases in the levels of these proteins. The reduction in the levels of phospho-PEA-15 proteins indicates the inhibition of anti-apoptotic function of PEA-15. Together, in vivo and in vitro results suggest that melatonin protects neurons against ischemic injury by maintaining levels of phospho-PEA-15 proteins.

Characterization of a MAPK Scaffolding Protein Logic Gate in Gonadotropes

In the pituitary gonadotropes, both protein kinase C (PKC) and MAPK/ERK signaling cascades are activated by GnRH. Phosphoprotein-enriched in astrocytes 15 (PEA-15) is a cytosolic ERK scaffolding protein, which is expressed in LβT2 gonadotrope cells. Pharmacological inhibition of PKC and small interfering RNA-mediated silencing of Gαq/11 revealed that GnRH induces accumulation of phosphorylated PEA-15 in a PKC-dependent manner. To investigate the potential role of PEA-15 in GnRH signaling, we examined the regulation of ERK subcellular localization and the activation of ribosomal S6 kinase, a substrate of ERK. Results obtained by cellular fractionation/Western blot analysis and immunohistochemistry revealed that GnRH-induced accumulation of phosphorylated ERK in the nucleus was attenuated when PEA-15 expression was reduced. Conversely, in the absence of GnRH stimulation, PEA-15 anchors ERK in the cytosol. Our data suggest that GnRH-induced nuclear translocation of ERK requires its release from PEA-15, which occurs upon PEA-15 phosphorylation by PKC. Additional gene-silencing experiments in GnRH-stimulated cells demonstrated that ribosomal S6 kinase activation was dependent on both PEA-15 and PKC. Furthermore, small interfering RNA-mediated knockdown of PEA-15 caused a reduction in GnRH-stimulated expression of early response genes Egr2 and c-Jun, as well as gonadotropin FSHβ-subunit gene expression. PEA-15 knockdown increased LHβ and common α-glycoprotein subunit mRNAs, suggesting a possible role in differential regulation of gonadotropin subunit gene expression. We propose that PEA-15 represents a novel point of convergence of the PKC and MAPK/ERK pathways under GnRH stimulation. PKC, ERK, and PEA-15 form an AND logic gate that shapes the response of the gonadotrope cell to GnRH.

Gingko bilobaExtract (EGb 761) Attenuates the Focal Cerebral Ischemic Injury-Induced Decrease in Astrocytic Phosphoprotein PEA-15 Levels

EGb 761 is an extract of Gingko biloba that is neuroprotective against focal cerebral ischemic injury. PEA-15 (phosphoprotein enriched in astrocytes 15) modulates cell proliferation and apoptosis. In this study, we investigated whether EGb 761 regulates the expression of PEA-15 and two phosphorylated forms of PEA-15 (Ser 104 and Ser 116) in middle cerebral artery occlusion (MCAO)-induced injury. Adult male rats were treated with vehicle or EGb 761 (100 mg/kg) prior to MCAO and cerebral cortices were collected 24 h after MCAO. A reduction in expression of PEA-15 and its phosphorylated forms induced by MCAO injury was detected using a proteomic approach. EGb 761 pretreatment prevented the ischemic injury-induced decrease in PEA-15 expression. Western blot analysis demonstrated that EGb 761 attenuates the injury-induced reduction in PEA-15, phospho-PEA-15 (Ser 104), phospho-PEA-15 (Ser 116). Phosphorylation of PEA-15 influences its anti-apoptotic function; a decrease in PEA-15 phosphorylation induces apoptotic cell death. The maintenance of PEA-15 phosphorylation by EGb 761 pretreatment during cerebral ischemic injury indicates that EGb 761 is a neuroprotective against cerebral ischemic injury.

ERK MAP kinase is targeted to RSK2 by the phosphoprotein PEA-15

The ERK pathway responds to extracellular stimuli and oncogenes by modulating cellular processes, including transcription, adhesion, survival, and proliferation. ERK has diverse substrates that carry out these functions. The processes that are modulated are determined in part by the substrates that ERK phosphorylates. We demonstrate that PEA-15 (phosphoprotein enriched in astrocytes, 15 kDa) targets ERK to RSK2 and thereby enhances RSK2 activation. PEA-15 independently bound ERK and RSK2 and increased ERK association with RSK2 in a concentration-dependent manner. PEA-15 increased RSK2 activity and CREB-mediated transcription, and this process was regulated by phosphorylation of PEA-15. Finally, phorbol ester stimulation of PEA-15-null lymphocytes resulted in impaired RSK2 activation that was rescued by exogenous PEA-15 expression. Therefore, PEA-15 functions as a scaffold to enhance ERK activation of RSK2, and this activity is regulated by phosphorylation. Thus, PEA-15 can integrate signal transduction to provide a specific physiological outcome from activation of the multipotent ERK MAP kinase pathway.

The PEA15 gene is overexpressed and related to insulin resistance in healthy first-degree relatives of patients with type 2 diabetes

Overexpression of the gene encoding phosphoprotein enriched in astrocytes 15 (PEA15), also known as phosphoprotein enriched in diabetes (PED), causes insulin resistance and diabetes in transgenic mice and has been observed in type 2 diabetic individuals. The aim of this study was to investigate whether PEA15 overexpression occurs in individuals at high risk of diabetes and whether it is associated with specific type 2 diabetes subphenotypes. We analysed PEA15 expression in euglycaemic first-degree relatives (FDR) of type 2 diabetic subjects. The expression of PEA15 in peripheral blood leucocytes (PBLs) paralleled that in fat and skeletal muscle tissues. In PBLs from the FDR, PEA15 expression was two-fold higher than in euglycaemic individuals with no family history of diabetes (control subjects), both at the protein and the mRNA level (p < 0.001). The expression of PEA15 was comparable in FDR and type 2 diabetic subjects and in each group close to one-third of the subjects expressed PEA15 levels more than 2 SD higher than the mean of control subjects. Subjects with IFG with at least one type 2 diabetes-affected FDR also overexpressed PEA15 (p < 0.05). In all the groups analysed, PEA15 expression was independent of sex and unrelated to age, BMI, waist circumference, systolic and diastolic BP, and fasting cholesterol, triacylglycerol and glucose levels. However, in euglycaemic FDR of type 2 diabetic subjects, PEA15 expression was inversely correlated with insulin sensitivity (r = -557, p = 0.01). We conclude that PEA15 overexpression represents a common defect in FDR of patients with type 2 diabetes and is correlated with reduced insulin sensitivity in these individuals.

RSK2 Activity Is Regulated by Its Interaction with PEA-15

The ERK MAP (mitogen-activated protein) kinase cascade modulates many cellular processes including transcription, adhesion, growth, survival, and proliferation. One target substrate of ERK involved in regulating transcription is the p90 ribosomal S6 kinase (RSK) isozyme, RSK2. Here we demonstrate that a small death effector domain-containing protein called PEA-15 binds RSK2. RSK2 and PEA-15 (phosphoprotein enriched in astrocytes, 15 kDa) co-precipitated from cells and were colocalized in the cytoplasm. Furthermore, purified PEA-15 bound in vitro translated RSK2, suggesting that these proteins interact directly. PEA-15 does not bind to RSK1 and therefore exhibits some binding specificity. RSK2 binds the COOH terminus of PEA-15 and does not interact with its NH2-terminal death effector domain. We show that this interaction has functional consequences including the inhibition of RSK2-dependent CREB transcription. PEA-15 expression also blocks histone H3 phosphorylation, an RSK2-dependent event that may contribute to effects on gene expression. These results can be attributed to two effects of PEA-15 on RSK2. First, PEA-15 blocks nuclear accumulation of RSK2 after epidermal growth factor stimulation. Second, PEA-15 inhibits RSK2 kinase activity by 50%. A mutant of PEA-15 that binds RSK2 but is localized to the nucleus had no effect on RSK2-dependent transcription. Interestingly, this mutant also did not affect RSK2 kinase activity. This may indicate that cytoplasmic retention of RSK2 is also required for PEA-15 to impair kinase activity. PEA-15 does not alter ERK phosphorylation of RSK2 and is not itself a substrate of RSK2. Hence the effects of PEA-15 on RSK2 represent a novel mechanism for the regulation of RSK2-mediated signaling.