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Patricia T.w. Cohen - One of the best experts on this subject based on the ideXlab platform.
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purification of Protein phosphatase 4 catalytic subunit inhibition by the antitumour drug fostriecin and other tumour suppressors and promoters
FEBS Letters, 1998Co-Authors: James C Hastie, Patricia T.w. CohenAbstract:Protein phosphatase 4 (PP4) is a Protein Serine/threonine phosphatase that predominantly localises to centrosomes and plays a role in microtubule organisation at centrosomes. Here, PP4 catalytic subunit has been purified from porcine testis to near homogeneity and a specific activity of 680 mU/mg against phosphorylase a. The antitumour drug, fostriecin, inhibits PP4 catalytic subunit (IC50 3 nM) with similar potency to PP2A catalytic subunit (IC50 1.5 nM). PP4 is also inhibited in the nanomolar range by several naturally occurring tumour promoters and toxins, with similar IC50 values to those obtained for PP2A. The gene for human PP4 catalytic subunit localises to 16p11.2.
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novel Protein Serine threonine phosphatases variety is the spice of life
Trends in Biochemical Sciences, 1997Co-Authors: Patricia T.w. CohenAbstract:Abstract The dephosphorylation of Proteins on Serine and threonine residues is a major mechanism of cellular regulation. Many novel Protein Serine/threonine phosphatases in the PPP family have recently been discovered and the insights that have been gained into their different functions are summarised in this review.
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the cyanobacterial toxin microcystin binds covalently to cysteine 273 on Protein phosphatase 1
FEBS Letters, 1995Co-Authors: Robert W Mackintosh, Patricia T.w. Cohen, Kevin N Dalby, David G. Campbell, Carol MackintoshAbstract:Abstract The interaction between Protein phosphatase 1 (PP1) and microcystin (MC) was stable in 1% SDS or 70% formic acid indicative of a covalent interaction. Here we isolate the MC-binding peptide and demonstrate that Cys273 of PP1 binds covalently to the methyl-dehydroalanine (Mdha) residue of the toxin. Mutation of Cys273 to Ala, Ser or leu abolished covalent binding to MC, as did reduction of the Mdha residue of the toxin with ethanethiol. The abolition of covalent binding increased the IC50 for toxin inhibition of PP1 by 5- to 20-fold. The covalent binding of MC to Protein Serine/threonine phosphatases explains the failure to detect this toxin post-mortem in suspected cases of MC poisoning.
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a novel human Protein Serine threonine phosphatase which possesses four tetratricopeptide repeat motifs and localizes to the nucleus
The EMBO Journal, 1994Co-Authors: Mao Xiang Chen, A. E. Mcpartlin, L. Brown, Yu Hua Chen, H. M. Barker, Patricia T.w. CohenAbstract:A novel human Protein Serine/threonine phosphatase, PP5, and a structurally related phosphatase in Saccharomyces cerevisiae, PPT1, have been identified from their cDNA and gene respectively. Their predicted molecular mass is 58 kDa and they comprise a C-terminal phosphatase catalytic domain and an N-terminal domain, which has four repeats of 34 amino acids, three of which are tandemly arranged. The phosphatase domain possesses all the invariant motifs of the PP1/PP2A/PP2B gene family, but is not closely related to any other known member (< or = 40% identity). Thus PP5 and PPT1 comprise a new subfamily. The repeats in the N-terminal domain are similar to the tetratricopeptide repeat (TPR) motifs which have been found in several Proteins that are required for mitosis, transcription and RNA splicing. Bacterially expressed PP5 is able to dephosphorylate Serine residues in Proteins and is more sensitive than PP1 to the tumour promoter okadaic acid. A 2.3 kb mRNA encoding PP5 is present in all human tissues examined. Investigation of the intracellular distribution of PP5 by immunofluorescence, using two different antibodies raised against the TPR and phosphatase domains, localizes PP5 predominantly to the nucleus. This suggests that, like other nuclear TPR-containing Proteins, it may play a role in the regulation of RNA biogenesis and/or mitosis.
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ppx a novel Protein Serine threonine phosphatase localized to centrosomes
The EMBO Journal, 1993Co-Authors: Neil D Brewis, Alasdair J Street, Alan R Prescott, Patricia T.w. CohenAbstract:Abstract The amino acid sequence of a novel mammalian Protein phosphatase, termed PPX (and designated PPP4 in the human genome nomenclature), has been deduced from the cDNA and shown to be 65% identical to PP2A alpha and PP2A beta and 45% identical to PPI isoforms, the predicted molecular mass being 35 kDa. PPX was expressed in the baculovirus system. Its substrate specificity and sensitivity to the inhibitors, okadaic acid and microcystin, were similar (but not identical) to the catalytic subunit of PP2A. However, PPX did not bind the 65 kDa regulatory subunit of PP2A. The intracellular localization of PPX was investigated by immunofluorescence using two different antibodies raised against bacterially expressed PPX and a PPX-specific peptide. These showed that although PPX was distributed throughout the cytoplasm and the nucleus, intense staining occurred at centrosomes. The centrosomal staining was apparent in interphase and at all stages of mitosis, except telophase. In contrast, antibodies directed against bacterially expressed PP2A were not specifically localized to centrosomes. The human autoantibody #5051, which stains the pericentriolar material, colocalizes with PPX antibodies, suggesting that PPX may play a role in microtubule nucleation.
Robert Roskoski - One of the best experts on this subject based on the ideXlab platform.
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cyclin dependent Protein Serine threonine kinase inhibitors as anticancer drugs
Pharmacological Research, 2019Co-Authors: Robert RoskoskiAbstract:Abstract Cyclins and cyclin-dependent Protein kinases (CDKs) are important Proteins that are required for the regulation and expression of the large number of components necessary for the passage through the cell cycle. The concentrations of the CDKs are generally constant, but their activities are controlled by the oscillation of the cyclin levels during each cell cycle. Additional CDK family members play significant roles in a wide range of activities including gene transcription, metabolism, and neuronal function. In response to mitogenic stimuli, cells in the G1-phase of the cell cycle produce D type cyclins that activate CDK4/6. These activated enzymes catalyze the monophosphorylation of the retinoblastoma Protein. Subsequently, CDK2-cyclin E catalyzes the hyperphosphorylation of Rb that promotes the release and activation of the E2F transcription factor, which in turn lead to the biosynthesis of dozens of Proteins required for cell cycle progression. Consequently, cells pass the G1-restriction point and are committed to complete cell division in the absence of mitogenic stimulation. CDK2-cyclin A, CDK1-cyclin A, and CDK1-cyclin B are required for S-, G2-, and M-phase progression. A crucial mechanism in controlling cell cycle progression is the precise timing of more than 32,000 phosphorylation and dephosphorylation reactions catalyzed by a network of Protein kinases and phosphoProtein phosphatases as determined by mass spectrometry. Increased cyclin or CDK expression or decreased levels of endogenous CDK modulators/inhibitors such as INK4 or CIP/KIP have been observed in a wide variety of carcinomas, hematological malignancies, and sarcomas. The pathogenesis of neoplasms because of mutations in the CDKs are rare. Owing to their role in cell proliferation, CDKs represent natural targets for anticancer therapies. Palbociclib, ribociclib, and abemaciclib are FDA-approved CDK4/6 inhibitors used in the treatment of breast cancer. These drugs have IC50 values for CKD4/6 in the low nanomolar range. These inhibitors bind in the cleft between the N-terminal and C-terminal lobes of the CDKs and they inhibit ATP binding. Like ATP, these agents form hydrogen bonds with hinge residues that connect the small and large lobes of Protein kinases. Like the adenine base of ATP, these antagonists interact with catalytic spine residues CS6, CS7, and CS8. These and other CDK antagonists are in clinical trials for the treatment of a wide variety of malignancies. As inhibitors of the cell cycle, it is not surprising that one of their most common toxicities is myelosuppression with decreased neutrophil production.
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targeting oncogenic raf Protein Serine threonine kinases in human cancers
Pharmacological Research, 2018Co-Authors: Robert RoskoskiAbstract:The Ras-Raf-MEK-ERK signal transduction cascade is arguably the most important oncogenic pathway in human cancers. Ras-GTP promotes the formation of active homodimers or heterodimers of A-Raf, B-Raf, and C-Raf by an intricate process. These enzymes are Protein-Serine/threonine kinases that catalyze the phosphorylation and activation of MEK1 and MEK2 which, in turn, catalyze the phosphorylation and activation of ERK1 and ERK2. The latter catalyze the regulatory phosphorylation of dozens of cytosolic and nuclear Proteins. The X-ray crystal structure of B-Raf-MEK1 depicts a face-to-face dimer with interacting activation segments; B-Raf is in an active conformation and MEK1 is in an inactive conformation. Besides the four traditional components in the Ras-Raf-MEK-ERK signaling module, scaffolding Proteins such as Kinase Suppressor of Ras (KSR1/2) play an important role in this signaling cascade by functioning as a scaffold Protein. RAS mutations occur in about 30% of all human cancers. Moreover, BRAFV600E mutations occur in about 8% of all cancers making this the most prevalent oncogenic Protein kinase. Vemurafenib and dabrafenib are B-RafV600E inhibitors that were approved for the treatment of melanomas bearing the V600E mutation. Coupling MEK1/2 inhibitors with B-Raf inhibitors is more effective in treating such melanomas and dual therapy is now the standard of care. Vemurafenib and cobimetanib, dabrafenib and trametinib, and encorafenib plus binimetinib are the FDA-approved combinations for the treatment of BRAFV600E melanomas. Although such mutations occur in other neoplasms including thyroid, colorectal, and non-small cell lung cancers, these agents are not as effective in treating these non-melanoma neoplasms. Vemurafenib and dabrafenib produce the paradoxical activation of the MAP kinase pathway in wild type BRAF cells. The precise mechanism for this activation is unclear, but drug-induced Raf activating side-to-side dimerization appears to be an essential step. Although 63%-76% of all people with advanced melanoma with the BRAF V600E mutation derive clinical benefit from combination therapy, median progression-free survival lasts only about nine months and 90% of patients develop resistance within one year. The various secondary resistance mechanisms include NRAS or KRAS mutations (20%), BRAF splice variants (16%), BRAFV600E/K amplifications (13%), MEK1/2 mutations (7%), and non-MAP kinase pathway alterations (11%). Vemurafenib and dabrafenib bind to an inactive form of B-Raf (αC-helixout and DFG-Din) and are classified as type I½ inhibitors. LY3009120 and lifirafenib, which are in the early drug-development stage, bind to a different inactive form of B-Raf (DFG-Dout) and are classified as type II inhibitors. Besides targeting B-Raf and MEK Protein kinases, immunotherapies that include ipilimumab, pembrolizumab, and nivolumab have been FDA-approved for the treatment of melanomas. Current clinical trials are underway to determine the optimal usage of targeted and immunotherapies.
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erk1 2 map kinases structure function and regulation
Pharmacological Research, 2012Co-Authors: Robert RoskoskiAbstract:ERK1 and ERK2 are related Protein-Serine/threonine kinases that participate in the Ras-Raf-MEK-ERK signal transduction cascade. This cascade participates in the regulation of a large variety of processes including cell adhesion, cell cycle progression, cell migration, cell survival, differentiation, metabolism, proliferation, and transcription. MEK1/2 catalyze the phosphorylation of human ERK1/2 at Tyr204/187 and then Thr202/185. The phosphorylation of both tyrosine and threonine is required for enzyme activation. Whereas the Raf kinase and MEK families have narrow substrate specificity, ERK1/2 catalyze the phosphorylation of hundreds of cytoplasmic and nuclear substrates including regulatory molecules and transcription factors. ERK1/2 are proline-directed kinases that preferentially catalyze the phosphorylation of substrates containing a Pro-Xxx-Ser/Thr-Pro sequence. Besides this primary structure requirement, many ERK1/2 substrates possess a D-docking site, an F-docking site, or both. A variety of scaffold Proteins including KSR1/2, IQGAP1, MP1, β-Arrestin1/2 participate in the regulation of the ERK1/2 MAP kinase cascade. The regulatory dephosphorylation of ERK1/2 is mediated by Protein-tyrosine specific phosphatases, Protein-Serine/threonine phosphatases, and dual specificity phosphatases. The combination of kinases and phosphatases make the overall process reversible. The ERK1/2 catalyzed phosphorylation of nuclear transcription factors including those of Ets, Elk, and c-Fos represents an important function and requires the translocation of ERK1/2 into the nucleus by active and passive processes involving the nuclear pore. These transcription factors participate in the immediate early gene response. The activity of the Ras-Raf-MEK-ERK cascade is increased in about one-third of all human cancers, and inhibition of components of this cascade by targeted inhibitors represents an important anti-tumor strategy. Thus far, however, only inhibition of mutant B-Raf (Val600Glu) has been found to be therapeutically efficacious.
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raf Protein Serine threonine kinases structure and regulation
Biochemical and Biophysical Research Communications, 2010Co-Authors: Robert RoskoskiAbstract:A-RAF, B-RAF, and C-RAF are a family of three Protein-Serine/threonine kinases that participate in the RAS-RAF-MEK-ERK signal transduction cascade. This cascade participates in the regulation of a large variety of processes including apoptosis, cell cycle progression, differentiation, proliferation, and transformation to the cancerous state. RAS mutations occur in 15–30% of all human cancers, and B-RAF mutations occur in 30–60% of melanomas, 30–50% of thyroid cancers, and 5–20% of colorectal cancers. Activation of the RAF kinases requires their interaction with RAS-GTP along with dephosphorylation and also phosphorylation by SRC family Protein-tyrosine kinases and other Protein-Serine/threonine kinases. The formation of unique side-to-side RAF dimers is required for full kinase activity. RAF kinase inhibitors are effective in blocking MEK1/2 and ERK1/2 activation in cells containing the oncogenic B-RAF Val600Glu activating mutation. RAF kinase inhibitors lead to the paradoxical increase in RAF kinase activity in cells containing wild-type B-RAF and wild-type or activated mutant RAS. C-RAF plays a key role in this paradoxical increase in downstream MEK-ERK activation.
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src kinase regulation by phosphorylation and dephosphorylation
Biochemical and Biophysical Research Communications, 2005Co-Authors: Robert RoskoskiAbstract:Abstract Src and Src-family Protein-tyrosine kinases are regulatory Proteins that play key roles in cell differentiation, motility, proliferation, and survival. The initially described phosphorylation sites of Src include an activating phosphotyrosine 416 that results from autophosphorylation, and an inhibiting phosphotyrosine 527 that results from phosphorylation by C-terminal Src kinase (Csk) and Csk homologous kinase. Dephosphorylation of phosphotyrosine 527 increases Src kinase activity. Candidate phosphotyrosine 527 phosphatases include cytoplasmic PTP1B, Shp1 and Shp2, and transmembrane enzymes include CD45, PTPα, PTPe, and PTPλ. Dephosphorylation of phosphotyrosine 416 decreases Src kinase activity. Thus far PTP-BL, the mouse homologue of human PTP-BAS, has been shown to dephosphorylate phosphotyrosine 416 in a regulatory fashion. The platelet-derived growth factor receptor Protein-tyrosine kinase mediates the phosphorylation of Src Tyr138; this phosphorylation has no direct effect on Src kinase activity. The platelet-derived growth factor receptor and the ErbB2/HER2 growth factor receptor Protein-tyrosine kinases mediate the phosphorylation of Src Tyr213 and activation of Src kinase activity. Src kinase is also a substrate for Protein-Serine/threonine kinases including Protein kinase C (Ser12), Protein kinase A (Ser17), and CDK1/cdc2 (Thr34, Thr46, and Ser72). Of the three Protein-Serine/threonine kinases, only phosphorylation by CDK1/cdc2 has been demonstrated to increase Src kinase activity. Although considerable information on the phosphoProtein phosphatases that catalyze the hydrolysis of Src phosphotyrosine 527 is at hand, the nature of the phosphatases that mediate the hydrolysis of phosphotyrosine 138 and 213, and phosphoSerine and phosphothreonine residues has not been determined.
James R Woodgett - One of the best experts on this subject based on the ideXlab platform.
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mitogen inactivation of glycogen synthase kinase 3 beta in intact cells via Serine 9 phosphorylation
Biochemical Journal, 1994Co-Authors: V Stambolic, James R WoodgettAbstract:Glycogen synthase kinase-3 (GSK-3), a Protein-Serine kinase implicated in cell-fate determination and differentiation, phosphorylates several regulatory Proteins that are activated by dephosphorylation in response to hormones or growth factors. GSK-3 beta is phosphorylated in vitro at Serine 9 by p70 S6 kinase and p90rsk-1, resulting in its inhibition [Sutherland, Leighton, and Cohen (1993) Biochem. J. 296, 15-19]. Using HeLa cells expressing GSK-3 beta or a mutant containing alanine at residue 9, we demonstrate that Serine 9 is modified in intact cells and is targeted specifically by p90rsk-1, and that phosphorylation leads to loss of activity. Since p90rsk-1 is directly activated by mitogen-activated Protein kinases, agonists of this pathway, such as insulin, repress GSK-3 function.
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modulation of the glycogen synthase kinase 3 family by tyrosine phosphorylation
The EMBO Journal, 1993Co-Authors: Kenneth Hughes, Eleni Nikolakaki, Simon E Plyte, N F Totty, James R WoodgettAbstract:Abstract Glycogen synthase kinase-3 (GSK-3) is a Protein Serine kinase implicated in the cellular response to insulin. The enzyme is the mammalian homologue of the zeste-white3 (shaggy) homeotic gene of Drosophila melanogaster and has been implicated in the regulation of the c-Jun/AP-1 transcription factor. In mammals this Protein Serine kinase is encoded by two related genes termed GSK-3 alpha and beta. Here, we demonstrate that these two Proteins and the fruit fly Protein are phosphorylated on tyrosine in vivo. Moreover, GSK-3 beta activity and function are shown to be dependent on tyrosine phosphorylation. The modified tyrosine residue is conserved in all members of the GSK-3 family and is equivalent to that required for activity by mitogen-activated Protein (MAP) kinases. However, unlike MAP kinases, GSK-3 is highly phosphorylated on tyrosine and thus active in resting cells.
Carol Mackintosh - One of the best experts on this subject based on the ideXlab platform.
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regulation of cytosolic enzymes in primary metabolism by reversible Protein phosphorylation
Current Opinion in Plant Biology, 1998Co-Authors: Carol MackintoshAbstract:Abstract Recent discoveries have revealed that cytosolic enzymes of sugar, amino acid, and isoprenoid synthesis, sucrose breakdown and the plasma membrane H+-ATPase are regulated by reversible Protein (Serine/threonine) phosphorylation. In some cases, phosphorylation creates a phosphopeptide motif that is recognized by and binds to 14-3-3 Proteins, and 14-3-3 binding changes the activity of the enzyme or ion pump. Intriguing new clues hint at how these cytosolic regulatory networks might link to signalling pathways triggered by hormones, nutrients, stresses, circadian rhythms, and other factors that regulate the Growth and development of the whole plant.
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the cyanobacterial toxin microcystin binds covalently to cysteine 273 on Protein phosphatase 1
FEBS Letters, 1995Co-Authors: Robert W Mackintosh, Patricia T.w. Cohen, Kevin N Dalby, David G. Campbell, Carol MackintoshAbstract:Abstract The interaction between Protein phosphatase 1 (PP1) and microcystin (MC) was stable in 1% SDS or 70% formic acid indicative of a covalent interaction. Here we isolate the MC-binding peptide and demonstrate that Cys273 of PP1 binds covalently to the methyl-dehydroalanine (Mdha) residue of the toxin. Mutation of Cys273 to Ala, Ser or leu abolished covalent binding to MC, as did reduction of the Mdha residue of the toxin with ethanethiol. The abolition of covalent binding increased the IC50 for toxin inhibition of PP1 by 5- to 20-fold. The covalent binding of MC to Protein Serine/threonine phosphatases explains the failure to detect this toxin post-mortem in suspected cases of MC poisoning.
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purification of type 1 Protein Serine threonine phosphatases by microcystin sepharose affinity chromatography
FEBS Letters, 1994Co-Authors: Greg Moorhead, Robert W Mackintosh, Nick Morrice, Timothy Gallagher, Carol MackintoshAbstract:A microcystin (MC)-Sepharose column was prepared by addition of 2-aminoethanethiol to the alpha, beta-unsaturated carbonyl of the N-methyldehydroalanine residue of MC-LR, followed by reaction of the introduced amino group with N-hydroxysuccinimide-activated CH-Sepharose. The MC-Sepharose bound Protein phosphatase-1 (PP1) with high capacity and purified human PP1 gamma in one step from E. coli extracts. It was also used to purify forms of PP1 bound to myofibrils from skeletal muscle. Two of these comprised PP1 complexed to N-terminal fragments of the M-subunit which enhance its myosin phosphatase activity, while the third comprised PP1 and an N-terminal fragment of the glycogen-binding (G)-subunit.
Hyuna Seong - One of the best experts on this subject based on the ideXlab platform.
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Murine Protein Serine-threonine kinase 38 activates p53 function through Ser15 phosphorylation.
Journal of Biological Chemistry, 2012Co-Authors: Hyuna Seong, Hyunjung HaAbstract:Murine Protein Serine-threonine kinase 38 (MPK38) is a member of the AMP-activated Protein kinase-related Serine/threonine kinase family. In this study, we show that MPK38 physically associates with p53 via the carboxyl-terminal domain of MPK38 and the central DNA-binding domain of p53. This interaction is increased by 5-fluorouracil or doxorubicin treatment and is responsible for Ser15 phosphorylation of p53. Ectopic expression of wild-type Mpk38, but not kinase-dead Mpk38, stimulates p53-mediated transcription in a dose-dependent manner and up-regulates p53 targets, including p53, p21, MDM2, and BAX. Consistently, knockdown of MPK38 shows an opposite trend, inhibiting p53-mediated transcription. MPK38 functionally enhances p53-mediated apoptosis and cell cycle arrest in a kinase-dependent manner by stimulating p53 nuclear translocation. We also demonstrate that MPK38-mediated p53 activation is induced by removing MDM2, a negative regulator of p53, from the p53-MDM2 complex as well as by stabilization of interaction between p53 and its positive regulators, including NM23-H1, Serine/threonine kinase receptor-associated Protein, and 14-3-3. This leads to the enhancement of p53 stability. Together, these results suggest that MPK38 may act as a novel regulator for promoting p53 activity through direct phosphorylation of p53 at Ser15.
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murine Protein Serine threonine kinase 38 stimulates tgf β signaling in a kinase dependent manner via direct phosphorylation of smad Proteins
Journal of Biological Chemistry, 2010Co-Authors: Hyuna Seong, Haiyoung JungAbstract:The present study demonstrated that murine Protein Serine/threonine kinase 38 (MPK38) coimmunoprecipitates with Smad Proteins (Smad2, -3, -4, and -7) and that this association is mediated by the catalytic kinase domain of MPK38. The association between MPK38 and Smad2, -3, and -4 was significantly increased by TGF-β or ASK1 signals, whereas these signals decreased association of MPK38 with Smad7. MPK38 stimulated TGF-β-induced transcription required for TGF-β-mediated biological functions, such as apoptosis and cell growth arrest, in a kinase-dependent manner. Knockdown of endogenous MPK38 showed an opposite effect, inhibiting TGF-β signaling. MPK38-mediated phosphorylation of Smad Proteins (Ser245 of Smad2, Ser204 of Smad3, Ser343 of Smad4, and Thr96 of Smad7) was also found to be crucial to the positive regulation of TGF-β signaling induced by MPK38. In addition, MPK38 enhanced nuclear translocation of Smad3, as well as redistribution of Smad7 from the nucleus to the cytoplasm, in response to TGF-β. Together, these results indicate that MPK38 functions as a stimulator of TGF-β signaling through direct interaction with and phosphorylation of Smad Proteins.
