Primary mucosa-associated lymphoid tissue lymphoma occurring in the rectum.
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Amoxicillin↗
Biomedical subjects
Philip Cohen
Publications and source records attributed to Philip Cohen.
Exploitation of KESTREL to identify NDRG family members as physiological substrates for SGK1 and GSK3.
We detected a protein in rabbit skeletal muscle extracts that was phosphorylated rapidly by SGK1 (serum- and glucocorticoid-induced kinase 1), but not by protein kinase Ba, and identified it as NDRG2 (N-myc downstream-regulated gene 2). SGK1 phosphorylated NDRG2 at Thr330, Ser332 and Thr348 in vitro. All three residues were phosphorylated in skeletal muscle from wild-type mice, but not from mice that do not express SGK1. SGK1 also phosphorylated the related NDRG1 isoform at Thr328, Ser330 and Thr346 (equivalent to Thr330, Ser332 and Thr348 of NDRG2), as well as Thr356 and Thr366. Residues Thr346, Thr356 and Thr366 are located within identical decapeptide sequences GTRSRSHTSE, repeated three times in NDRG1. These threonines were phosphorylated in NDRG1 in the liver, lung, spleen and skeletal muscle of wild-type mice, but not in SGK1-/- mice. Knock-down of SGK1 in HeLa cells using small interfering RNA also suppressed phosphorylation of the threonine residues in the repeat region of NDRG1. The phosphorylation of NDRG1 by SGK1 transformed it into an excellent substrate for GSK3 (glycogen synthase kinase 3), which could then phosphorylate Ser342, Ser352 and Ser362 in the repeat region. Incubation of HeLa cells with the specific GSK3 inhibitor CT 99021 increased the electrophoretic mobility of NDRG1 in HeLa cells, demonstrating that this protein is phosphorylated by GSK3 in cells. Our results identify NDRG1 and NDRG2 as physiological substrates for SGK1, and demonstrate that phosphorylation of NDRG1 by SGK1 primes it for phosphorylation by GSK3.
Amino Acid Sequence↗
Identification of filamin C as a new physiological substrate of PKBalpha using KESTREL.
We detected a protein in rabbit skeletal muscle extracts that was phosphorylated rapidly by PKBa (protein kinase Ba), but not by SGK1 (serum- and glucocorticoid-induced kinase 1), and identified it as the cytoskeletal protein FLNc (filamin C). PKBa phosphorylated FLNc at Ser2213 in vitro, which lies in an insert not present in the FLNa and FLNb isoforms. Ser2213 became phosphorylated when C2C12 myoblasts were stimulated with insulin or epidermal growth factor, and phosphorylation was prevented by low concentrations of wortmannin, at which it is a relatively specific inhibitor of phosphoinositide 3-kinase. PD 184352 [an inhibitor of the classical MAPK (mitogen-activated protein kinase) cascade] and/or rapamycin [an inhibitor of mTOR (mammalian target of rapamycin)] had no effect. Insulin also induced the phosphorylation of FLNc at Ser2213 in cardiac muscle in vivo, but not in cardiac muscle that does not express PDK1 (3-phosphoinositide-dependent kinase 1), the upstream activator of PKB. These results identify the muscle-specific isoform FLNc as a new physiological substrate for PKB.
3-Phosphoinositide-Dependent Protein Kinases↗
A novel UBA and UBX domain protein that binds polyubiquitin and VCP and is a substrate for SAPKs.
A widely expressed protein containing UBA (ubiquitin-associated) and UBX (ubiquitin-like) domains was identified as a substrate of SAPKs (stress-activated protein kinases). Termed SAKS1 (SAPK substrate-1), it was phosphorylated efficiently at Ser200 in vitro by SAPK3/p38gamma, SAPK4/p38delta and JNK (c-Jun N-terminal kinase), but weakly by SAPK2a/p38alpha, SAPK2b/p38beta2 or ERK (extracellular-signal-regulated kinase) 2. Ser200, situated immediately N-terminal to the UBX domain, became phosphorylated in HEK-293 (human embryonic kidney) cells in response to stressors. Phosphorylation was not prevented by SB 203580 (an inhibitor of SAPK2a/p38alpha and SAPK2b/p38beta2) and/or PD 184352 (which inhibits the activation of ERK1 and ERK2), and was similar in fibroblasts lacking both SAPK3/p38gamma and SAPK4/p38delta or JNK1 and JNK2. SAKS1 bound ubiquitin tetramers and VCP (valosin-containing protein) in vitro via the UBA and UBX domains respectively. The amount of VCP in cell extracts that bound to immobilized GST (glutathione S-transferase)-SAKS1 was enhanced by elevating the level of polyubiquitinated proteins, while SAKS1 and VCP in extracts were coimmunoprecipitated with an antibody raised against S5a, a component of the 19 S proteasomal subunit that binds polyubiquitinated proteins. PNGase (peptide N-glycanase) formed a 1:1 complex with VCP and, for this reason, also bound to immobilized GST-SAKS1. We suggest that SAKS1 may be an adaptor that directs VCP to polyubiquitinated proteins, and PNGase to misfolded glycoproteins, facilitating their destruction by the proteasome.
Adenosine Triphosphatases↗
Signalling pathways involved in multisite phosphorylation of the transcription factor ATF-2.
The multisite phosphorylation of the transcription factor ATF-2 was investigated using transformed embryonic fibroblasts from wild-type mice and mice deficient in c-Jun N-terminal kinases (JNK)1 and 2, and in the presence and absence of inhibitors of p38 mitogen-activated protein kinase (p38 MAPK) and the classical MAP kinase cascade. In wild-type cells, p38 MAPK and extracellular signal-regulated protein kinase (ERK)1/2 were not rate limiting for the phosphorylation of Thr69, Thr71 or Ser90. In JNK-deficient cells, p38 MAPK substituted for JNK partially in the phosphorylation of Thr69 and p38 MAPK or ERK1/2 in the phosphorylation of Thr71. JNK was the only MAP kinase that phosphorylated Ser90 under the conditions examined.
Activating Transcription Factor 2↗
Philip Cohen.
Philip Cohen trained at University College London and, after postdoctoral research at the University of Washington, joined the University of Dundee Scotland, in 1971, where he has worked ever since. He is a Royal Society Research Professor and Director of the Medical Research Council Protein Phosphorylation Unit. His main contributions have been in the area of protein phosphorylation and its role in cell regulation and human disease. In 1998, he was knighted for his contributions to biochemistry and the development of Life Sciences at Dundee.
Career Choice↗
TAB3, a new binding partner of the protein kinase TAK1.
We have identified a new binding partner of the TGFbeta (transforming growth factor-beta)-activated protein kinase (TAK1), termed TAB3 (TAK1-binding protein-3), which shares 48% amino acid sequence identity with TAB2. Our results indicate that two distinct TAK1 complexes are present in cells. One comprises TAK1 complexed with TAB1 and TAB2, and the other TAK1 complexed with TAB1 and TAB3. Both complexes are activated in response to tumour necrosis factor-alpha or interleukin-1 in human epithelial KB cells or bacterial lipopolysaccharide in RAW264.7 macrophages, and are subject to feedback control by stress-activated protein kinase 2a (SAPK2a; also called p38alpha). The electrophoretic mobility of TAB2 and TAB3 decreases in response to these agonists or osmotic shock, and is reversed by treatment with protein phosphatase-1. The decrease in mobility of TAB3 is prevented if the cells are incubated with SB 203580 before stimulation, but treatment with SB 203580 produces forms of TAB2 with a mobility intermediate between that observed for TAB2 in unstimulated and stimulated cells. Similar results were obtained in embryonic fibroblasts from mice deficient in SAPK2a/p38alpha. Our results indicate that TAB3 is phosphorylated via the SAPK2a/p38alpha pathway, whereas TAB2 is phosphorylated at two or more sites by both an SAPK2a/p38alpha-dependent and an SB 203580-independent kinase. The SAPK2a/p38alpha-mediated phosphorylation of TAB2 and TAB3 may contribute to the SAPK2a/p38alpha-mediated feedback control of TAK1 activity that also involves the phosphorylation of TAB1. We also show that the agonist-induced activation of TAK1 complexes requires the phosphorylation of the TAK1 catalytic subunit at a serine/threonine residue(s).
Adaptor Proteins, Signal Transducing↗
HSJ people. Off the scale.
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Health Care Surveys↗
Stimulation of renal Na+ dicarboxylate cotransporter 1 by Na+/H+ exchanger regulating factor 2, serum and glucocorticoid inducible kinase isoforms, and protein kinase B.
Renal tubular citrate transport is accomplished by electrogenic Na(+) coupled dicarboxylate transporter NaDC-1, a carrier subjected to regulation by acidosis. Trafficking of the Na(+)/H(+) exchanger NHE3 is controlled by NHE regulating factors NHERF-1 and NHERF-2 and the serum and glucocorticoid inducible kinase SGK1. To test for a possible involvement in NaDC-1 regulation, mRNA encoding NaDC-1 was injected into Xenopus oocytes with or without cRNA encoding NHERF-1, NHERF-2, SGK1, SGK2, SGK3, and/or the constitutively active form of the related protein kinase B ((T308,S473D)PKB). Succinate induced inward currents (I(succ)) were taken as a measure of transport rate. Coexpression of neither NHERF-1 nor NHERF-2 in NaDC-1 expressing oocytes significantly altered I(succ). On the other hand, coexpression of SGK1, SGK3, and (T308,S473D)PKB stimulated I(succ), an effect further stimulated by additional coexpression of NHERF-2 but not of NHERF-1. The action of the kinases and NHERF-2 may link urinary citrate excretion to proximal tubular H(+) secretion.
Animals↗
Further evidence that the tyrosine phosphorylation of glycogen synthase kinase-3 (GSK3) in mammalian cells is an autophosphorylation event.
Phosphorylation of the endogenous GSK3alpha (glycogen synthase kinase-3alpha) at Tyr279 and GSK3beta at Tyr216 was suppressed in HEK-293 or SH-SY5Y cells by incubation with pharmacological inhibitors of GSK3, but not by an Src-family inhibitor, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4- d ]pyrimidine (PP2), or a general protein tyrosine kinase inhibitor (genistein). GSK3beta transfected into HEK-293 cells or Escherichia coli became phosphorylated at Tyr216, but catalytically inactive mutants did not. GSK3beta expressed in insect Sf 21 cells or E. coli was extensively phosphorylated at Tyr216, but the few molecules lacking phosphate at this position could autophosphorylate at Tyr216 in vitro after incubation with MgATP. The rate of autophosphorylation was unaffected by dilution and was suppressed by the GSK3 inhibitor kenpaullone. Wild-type GSK3beta was unable to catalyse the tyrosine phosphorylation of catalytically inactive GSK3beta lacking phosphate at Tyr216. Our results indicate that the tyrosine phosphorylation of GSK3 is an intramolecular autophosphorylation event in the cells that we have studied and that this modification enhances the stability of the enzyme.
Cell Line↗
Monoclonal gammopathy in extranodal marginal zone lymphoma (ENMZL) correlates with advanced disease and bone marrow involvement.
Monoclonal gammopathy is a well-recognized occurrence in splenic marginal zone lymphoma (MZL); however, its prevalence has never been reported in extranodal MZL (ENMZL). We present results of a retrospective analysis of 26 patients with newly diagnosed as well as previously treated ENMZL. Monoclonal gammopathy was detected in 7 out of 26 (27%) patients. All patients with a monoclonal gammopathy had stage IV disease, and most of them had involvement of lymph nodes and/or bone marrow (n = 6). Only 1 out of 7 patients with a monoclonal spike had stage IV disease diagnosed based on multifocal mucosal involvement. There was also a strong correlation between the involvement of bone marrow and the presence of a monoclonal spike (Fisher's exact test, P = 0.0007). Prevalence of monoclonal gammopathy is higher than previously recognized and indicates advanced disease. However, the prognostic significance of the presence of monoclonal gammopathy in this population is unknown.
Adult↗
GSK3 inhibitors: development and therapeutic potential.
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Animals↗
D4476, a cell-permeant inhibitor of CK1, suppresses the site-specific phosphorylation and nuclear exclusion of FOXO1a.
The protein kinase CK1 phosphorylates serine residues that are located close to another phosphoserine in the consensus pSer-Xaa-Xaa-Ser. This specificity generates regions in its target proteins containing two or more neighbouring phosphoserine residues, termed here multisite phosphorylation domains (MPDs). In this paper, we demonstrate that D4476 is a potent and rather selective inhibitor of CK1 in vitro and in cells. In H4IIE hepatoma cells, D4476 specifically inhibits the phosphorylation of endogenous forkhead box transcription factor O1a (FOXO1a) on Ser322 and Ser325 within its MPD, without affecting the phosphorylation of other sites. Our results indicate that these residues are targeted by CK1 in vivo and that the CK1-mediated phosphorylation of the MPD is required for accelerated nuclear exclusion of FOXO1a in response to IGF-1 and insulin. D4476 is much more potent and specific than IC261 or CKI-7, and is therefore the most useful CK1 inhibitor currently available for identifying physiological substrates of CK1.
Benzamides↗
Phosphorylation of microtubule-associated protein tau by isoforms of c-Jun N-terminal kinase (JNK).
Microtubule-associated protein tau in a hyperphosphorylated state is the major component of the filamentous lesions that define a number of neurodegenerative diseases commonly referred to as tauopathies. Hyperphosphorylation of tau at most sites appears to precede filament assembly. Many of the hyperphosphorylated sites are serine/threonine-proline sequences. Here we show that c-Jun N-terminal kinases JNK1, JNK2 and JNK3 phosphorylate tau at many serine/threonine-prolines, as assessed by the generation of the epitopes of phosphorylation-dependent anti-tau antibodies. Of the three protein kinases, JNK2 phosphorylated the most sites in tau, followed by JNK3 and JNK1. Phosphorylation by JNK isoforms resulted in a greatly reduced ability of tau to promote microtubule assembly. These findings extend the number of candidate protein kinases for the hyperphosphorylation of tau in Alzheimer's disease and other neurodegenerative disorders.
Animals↗
ERF nuclear shuttling, a continuous monitor of Erk activity that links it to cell cycle progression.
The ets domain transcriptional repressor ERF is an effector of the receptor tyrosine kinase/Ras/Erk pathway, which, it has been suggested, is regulated by subcellular localization as a result of Erk-dependent phosphorylation and is capable of suppressing cell proliferation and ras-induced tumorigenicity. Here, we analyze the effect of ERF phosphorylation on nuclear import and export, the timing of its phosphorylation and dephosphorylation in relation to its subcellular location, Erk activity, and the requirements for ERF-induced cell cycle arrest. Our findings indicate that ERF continuously shuttles between the nucleus and the cytoplasm and that both phosphorylation and dephosphorylation of ERF occur within the nucleus. While nuclear import is not affected by phosphorylation, ERF nuclear export and cytoplasmic release require multisite phosphorylation and dephosphorylation. ERF export is CRM1 dependent, although ERF does not have a detectable nuclear export signal. ERF phosphorylation and export correlate with the levels of nuclear Erk activity. The cell cycle arrest induced by nonphosphorylated ERF requires the wild-type retinoblastoma protein and can be suppressed by overexpression of cyclin. These data suggest that ERF may be a very sensitive and constant sensor of Erk activity that can affect cell cycle progression through G(1), providing another link between the Ras/Erk pathway and cellular proliferation.
Animals↗
Regulation of the epithelial Ca2+ channel TRPV5 by the NHE regulating factor NHERF2 and the serum and glucocorticoid inducible kinase isoforms SGK1 and SGK3 expressed in Xenopus oocytes.
The epithelial Ca2+ channel TRPV5 (ECaC1) plays a key role in renal and intestinal Ca2+ (re)absorption and is thus regulated by 1,25(OH) 2D3. The present study aims to explore whether TRPV5 is regulated by the serum and glucocorticoid inducible kinase SGK1, a kinase transcriptionally upregulated by 1,25(OH) 2D3. To this end cRNA encoding TRPV5 has been injected into Xenopus oocytes with or without additional injection of SGK1, its isoforms SGK2 and SGK3, constitutively active (S422D)SGK1, inactive (K127N)SGK1, constitutively active (T308D,S473D)PKB and/or the Na+/H+ exchanger regulating factor NHERF2. In Xenopus laevisoocytes expression of TRPV5 increases uptake of tracer Ca(S422D;) and induces a Ca2+ current (ICa). In the presence of Cl-, TRPV5 mediated Ca2+ entry leads to secondary activation of Ca(2+)-sensitive Cl- channels (ICl(Ca)). Coexpression of TRPV5 with both (S422D)SGK1 and NHERF2 stimulates tracer Ca2+ entry, ICa and ICl(Ca). The effect of (S422D)SGK1 on TRPV5 and NHERF2 expressing oocytes is mimicked by SGK1 and SGK3, but not by SGK2, constitutively active (T308D,S473D)PKB or inactive (K127N)SGK1. The observations suggest that SGK1, SGK3 and NHERF2 regulate TRPV5 and are thus likely to participate in the regulation of calcium homeostasis.
Animals↗
Synthetic anisomycin analogues activating the JNK/SAPK1 and p38/SAPK2 pathways.
The synthesis of C(4)H and C(4)Me analogues of the JNK/p38 pathway activator anisomycin, based upon an aldol or Claisen construction of the C(3)-C(4) bond, has been demonstrated. The relative activation of the JNK/SAPK1 and p38/SAPK2 pathways in RAW macrophages by these analogues, and their synthetic precursors, has been assessed using immunoblot assays against phosphorylated c-Jun and MAPKAP-K2. These studies demonstrate that some of the synthetic C(4) analogues are also potent activators of these stress kinase pathways.
Animals↗
Feedback control of the protein kinase TAK1 by SAPK2a/p38alpha.
TAB1, a subunit of the kinase TAK1, was phosphorylated by SAPK2a/p38alpha at Ser423, Thr431 and Ser438 in vitro. TAB1 became phosphorylated at all three sites when cells were exposed to cellular stresses, or stimulated with tumour necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1) or lipopolysaccharide (LPS). The phosphorylation of Ser423 and Thr431 was prevented if cells were pre-incubated with SB 203580, while the phosphorylation of Ser438 was partially inhibited by PD 184352. Ser423 is the first residue phosphorylated by SAPK2a/p38alpha that is not followed by proline. The activation of TAK1 was enhanced by SB 203580 in LPS-stimulated macrophages, and by proinflammatory cytokines or osmotic shock in epithelial KB cells or embryonic fibroblasts. The activation of TAK1 by TNF-alpha, IL-1 or osmotic shock was also enhanced in embryonic fibroblasts from SAPK2a/p38alpha-deficient mice, while incubation of these cells with SB 203580 had no effect. Our results suggest that TAB1 participates in a SAPK2a/p38alpha-mediated feedback control of TAK1, which not only limits the activation of SAPK2a/p38alpha but synchronizes its activity with other signalling pathways that lie downstream of TAK1 (JNK and IKK).
Adaptor Proteins, Signal Transducing↗