Identification of the autophosphorylation sites and characterization of their effects in the protein kinase DYRK1A

2001 ◽  
Vol 359 (3) ◽  
pp. 497-505 ◽  
Author(s):  
Sunke HIMPEL ◽  
Pascal PANZER ◽  
Klaus EIRMBTER ◽  
Hanna CZAJKOWSKA ◽  
Muhammed SAYED ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Map Kinase ◽  
Mammalian Cells ◽  
Catalytic Domain ◽  
Molecular Model ◽  
Enzymic Activity ◽  
Mitogen Activated Protein Kinase ◽  
Kinase Family ◽  
Extracellular Signal ◽  
Mitogen Activated Protein

Protein kinases of the DYRK (‘dual-specificity tyrosine-regulated kinase’) family are characterized by a conserved Tyr-Xaa-Tyr motif (Tyr-319–Tyr-321) in a position exactly corresponding to the activation motif of the mitogen-activated protein kinase (MAP kinase) family (Thr-Xaa-Tyr). In a molecular model of the catalytic domain of DYRK1A, the orientation of phosphorylated Tyr-321 is strikingly similar to that of Tyr-185 in the known structure of the activated MAP kinase, extracellular-signal-regulated kinase 2. Consistent with our model, substitution of Tyr-321 but not of Tyr-319 by phenylalanine markedly reduced the enzymic activity of recombinant DYRK1A expressed in either Escherichia coli or mammalian cells. Direct identification of phosphorylated residues by tandem MS confirmed that Tyr-321, but not Tyr-319, was phosphorylated. When expressed in COS-7 cells, DYRK1A was found to be fully phosphorylated on Tyr-321. A catalytically inactive mutant of DYRK1A contained no detectable phosphotyrosine, indicating that Tyr-321 is autophosphorylated by DYRK1A. MS identified Tyr-111 and Ser-97 as additional autophosphorylation sites in the non-catalytic N-terminal domain of bacterially expressed DYRK1A. Enzymic activity was not affected in the DYRK1A-Y111F mutant. The present experimental data and the molecular model indicate that the activity of DYRK1A is dependent on the autophosphorylation of a conserved tyrosine residue in the activation loop.

scholarly journals Role of MLK3 in the Regulation of Mitogen-Activated Protein Kinase Signaling Cascades

2005 ◽  
Vol 25 (9) ◽  
pp. 3670-3681 ◽  
Author(s):  
Deborah Brancho ◽  
Juan-Jose Ventura ◽  
Anja Jaeschke ◽  
Beth Doran ◽  
Richard A. Flavell ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Map Kinase ◽  
Selective Reduction ◽  
P38 Map Kinase ◽  
Mitogen Activated Protein Kinase ◽  
Signaling Cascades ◽  
Extracellular Signal ◽  
Mitogen Activated Protein ◽  
Jnk Activation ◽  
Protein Kinase Signaling

ABSTRACT Mixed-lineage protein kinase 3 (MLK3) is a member of the mitogen-activated protein (MAP) kinase kinase kinase group that has been implicated in multiple signaling cascades, including the NF-κB pathway and the extracellular signal-regulated kinase, c-Jun NH2-terminal kinase (JNK), and p38 MAP kinase pathways. Here, we examined the effect of targeted disruption of the murine Mlk3 gene. Mlk3 −/− mice were found to be viable and healthy. Primary embryonic fibroblasts prepared from these mice exhibited no major signaling defects. However, we did find that MLK3 deficiency caused a selective reduction in tumor necrosis factor (TNF)-stimulated JNK activation. Together, these data demonstrate that MLK3 contributes to the TNF signaling pathway that activates JNK.

scholarly journals Selective deficiency in protein kinase C isoenzyme expression and inadequacy in mitogen-activated protein kinase activation in cord blood T cells

2003 ◽  
Vol 370 (2) ◽  
pp. 497-503 ◽  
Author(s):  
Charles S.T. HII ◽  
Maurizio COSTABILE ◽  
George C. MAYNE ◽  
Channing J. DER ◽  
Andrew W. MURRAY ◽  
...  
Keyword(s):  
T Cells ◽  
Protein Kinase ◽  
Cord Blood ◽  
Map Kinase ◽  
Map Kinases ◽  
Mitogen Activated Protein Kinase ◽  
Biochemical Basis ◽  
Kinase Activation ◽  
Extracellular Signal ◽  
Mitogen Activated Protein

The biochemical basis for the reduced lymphokine production by neonatal T cells compared with adult T cells remains poorly defined. Previous studies have raised the possibility that neonatal T cells could be deficient in their ability to transmit signals via protein kinase (PK) C. We now report that while PKC-dependent activation of the mitogen-activated protein (MAP) kinases, c-Jun N-terminal protein kinase and the extracellular signal-regulated protein kinase (ERK)1/ERK2, was deficient in cord blood T cells compared with adult blood T cells, marked activation of the MAP kinases in cord blood T cells was achieved via PKC-independent means. Consistent with a deficiency in the signalling capability of PKC, cord blood T cells were selectively deficient in the expression of PKCβI, ∊, θ and ζ. Stimulation of cord blood T cells resulted in a time-dependent increase in PKC expression, with increases detectable by 4h. This was accompanied by an enhancement in MAP kinase activation via PKC-dependent means. These novel data suggest that an inadequacy in PKC-MAP kinase signalling may be responsible, at least in part, for the phenotype of cord blood T cells.

α1-Adrenergic activation of myocardial Na-K-2Cl cotransport involving mitogen-activated protein kinase

1998 ◽  
Vol 275 (2) ◽  
pp. H641-H652 ◽  
Author(s):  
Geir Øystein Andersen ◽  
Mette Enger ◽  
G. Hege Thoresen ◽  
Tor Skomedal ◽  
Jan-Bjørn Osnes
Keyword(s):  
Protein Kinase ◽  
Map Kinase ◽  
Mitogen Activated Protein Kinase ◽  
Extracellular Signal ◽  
Rat Hearts ◽  
Protein Map ◽  
Mitogen Activated Protein ◽  
Β Blocker ◽  
Map Kinase Pathway ◽  
Erk Activity

The translocation mechanisms involved in the α1-adrenoceptor-stimulated efflux of the potassium analog86Rb+were studied in isolated rat hearts. Phenylephrine (in the presence of a β-blocker) increased the efflux of86Rb+and42K+, and the Na-K-2Cl (or K-Cl) cotransport inhibitor bumetanide reduced the response by 42 ± 11%. Furosemide inhibited the response with a lower potency than that of bumetanide. The bumetanide-insensitive efflux was largely sensitive to the K+ channel inhibitor 4-aminopyridine. Inhibitors of the Na+/H+exchanger or the Na+-K+pump had no effect on the increased86Rb+efflux. The activation of the Na-K-2Cl cotransporter was dependent on the extracellular signal-regulated kinase (ERK) subgroup of the mitogen-activated protein (MAP) kinase family. Phenylephrine stimulation increased ERK activity 3.4-fold. PD-98059, an inhibitor of the ERK cascade, reduced both the increased86Rb+efflux and ERK activity. Specific inhibitors of protein kinase C and Ca2+/calmodulin-dependent kinase II had no effect. In conclusion, α1-adrenoceptor stimulation increases86Rb+efflux from the rat heart via K+channels and a Na-K-2Cl cotransporter. Activation of the Na-K-2Cl cotransporter is apparently dependent on the MAP kinase pathway.

scholarly journals p63α and γ Induce TAU Phosphorylation in Cultured Mammalian Cells

2010 ◽  
Vol 4 ◽  
pp. JEN.S6295
Author(s):  
Claudie Hooper ◽  
Reem Soliman ◽  
Simon Lovestone ◽  
Richard Killick
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Protein Kinase ◽  
Mammalian Cells ◽  
Neuronal Development ◽  
Glycogen Synthase ◽  
Tau Phosphorylation ◽  
Mitogen Activated Protein Kinase ◽  
Extracellular Signal ◽  
Mitogen Activated Protein

Here we show by western blotting that transcriptionally active isoforms of p63 (p63α and p63γ) induce the phosphorylation of human 2N4R tau at the tau-1/AT8 epitope in HEK293a cells; a phospho-epitope increased in Alzheimer's disease. Confocal microscopy shows that tau and p63α are spatially separated intracellularly. Tau was found in the cytoskeletal compartment, whilst p63α was located in the nucleus, indicating that the effects of p63 on tau phosphorylation are indirectly mediated. Tau phosphorylation occurred independently of the known tau kinases, protein kinase C delta (PKCδ), c-Jun N-terminal kinase (JNK), extracellular-signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (p38), glycogen synthase kinase 3 (GSK3), v-akt murine thymoma viral oncogene homolog (AKT) and cyclin-dependent kinase 5 (Cdk5) and the tau protein phosphatases (PP), PP1 and PP2A-Aα/β. Considering that p63 and tau are both associated with developmental processes, these findings have ramifications for neuronal development and synaptic plasticity and also neurodegenerative diseases such as Alzheimer's disease and other tauopathies.

scholarly journals An analysis of the phosphorylation and activation of extracellular-signal-regulated protein kinase 5 (ERK5) by mitogen-activated protein kinase kinase 5 (MKK5) in vitro

2003 ◽  
Vol 372 (2) ◽  
pp. 567-575 ◽  
Author(s):  
Nimesh MODY ◽  
David G. CAMPBELL ◽  
Nick MORRICE ◽  
Mark PEGGIE ◽  
Philip COHEN
Keyword(s):  
Protein Kinase ◽  
Catalytic Domain ◽  
Similar Rate ◽  
Mitogen Activated Protein Kinase ◽  
Glutathione S Transferase ◽  
293 Cells ◽  
Extracellular Signal ◽  
Mitogen Activated Protein ◽  
Protein Kinase Kinase

MKK5 expressed as a glutathione S-transferase fusion protein in human embryonic kidney 293 cells activated full-length extracellular-signal-regulated protein kinase (ERK)5 (ERK5wt) as well as the isolated catalytic domain (ERK5cat) in vitro. Activation was accompanied by the phosphorylation of Thr219 and Tyr221, the former residue being phosphorylated preferentially. ERK5cat phosphorylated at Thr219, but not Tyr221, possessed 10% of the activity of the doubly phosphorylated protein towards myelin basic protein, whereas ERK5cat phosphorylated at Tyr221 alone was much less active. Activated ERK5 phosphorylated itself at a number of residues, including Thr28, Ser421, Ser433, Ser496, Ser731 and Thr733. ERK5 phosphorylated at Thr219, but not Tyr221, phosphorylated itself at a similar rate to ERK5 phosphorylated at both Thr219 and Tyr221. Activated ERK5 also phosphorylated mitogen-activated protein kinase kinase 5 (MKK5) extensively at Ser129, Ser137, Ser142 and Ser149, which are located within the region in MKK5 that is thought to interact with ERK5.

Effects of exercise and insulin on mitogen-activated protein kinase signaling pathways in rat skeletal muscle

1996 ◽  
Vol 271 (2) ◽  
pp. E403-E408 ◽  
Author(s):  
L. J. Goodyear ◽  
P. Y. Chang ◽  
D. J. Sherwood ◽  
S. D. Dufresne ◽  
D. E. Moller
Keyword(s):  
Skeletal Muscle ◽  
Protein Kinase ◽  
Map Kinase ◽  
Signaling Pathways ◽  
Mammalian Cells ◽  
Mitogen Activated Protein Kinase ◽  
Kinase Signaling ◽  
Rat Skeletal Muscle ◽  
Map Kinase Signaling ◽  
Mitogen Activated Protein

Studies in mammalian cells have established the existence of at least three distinct mitogen-activated protein kinase (MAP kinase) signaling pathways that are activated by a variety of growth factors and/or environmental stressors. We determined whether physical exercise, a physiological stressor, and insulin, a metabolic stimulator and growth factor, activate the c-jun NH2-terminus kinase (JNK), the p38 kinase, and/or the extracellular regulatory kinases (ERK; p42MAPK and p44MAPK) signaling pathways in rat skeletal muscle. Animals were studied immediately after running on a motorized treadmill for 10-60 min (20 m/min, 10% grade) or 5-30 min after an intraperitoneal injection of insulin (20 U/rat). Exercise increased skeletal muscle JNK activity by two- to threefold throughout the time course studied, whereas insulin did not significantly increase JNK activity. The p38 activity was slightly stimulated by exercise and not by insulin. The ERK kinase pathway, as assessed by ribosomal S6 kinase-2 activity assays and phosphospecific p42MAPK/p4NAPK immunoblotting, was stimulated by both exercise and insulin. These data are the first demonstration of exercise stimulating multiple intracellular signaling pathways in skeletal muscle. Activation of these MAP kinase signaling pathways may mediate changes in skeletal muscle growth and metabolism that occur in response to exercise.

scholarly journals Multiple regulatory domains on the Byr2 protein kinase.

1997 ◽  
Vol 17 (10) ◽  
pp. 5876-5887 ◽  
Author(s):  
H Tu ◽  
M Barr ◽  
D L Dong ◽  
M Wigler
Keyword(s):  
Protein Kinase ◽  
Catalytic Domain ◽  
Intramolecular Interaction ◽  
Mitogen Activated Protein Kinase ◽  
Regulatory Domain ◽  
Regulatory Domains ◽  
Kinase Catalytic Domain ◽  
Extracellular Signal ◽  
Mitogen Activated Protein ◽  
G Protein Alpha Subunit

Byr2 protein kinase, a homolog of mammalian mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEKK) and Saccharomyces cerevisiae STE11, is required for pheromone-induced sexual differentiation in the fission yeast Schizosaccharomyces pombe. Byr2 functions downstream of Ste4, Ras1, and the membrane-associated receptor-coupled heterotrimeric G-protein alpha subunit, Gpa1. Byr2 has a distinctive N-terminal kinase regulatory domain and a characteristic C-terminal kinase catalytic domain. Ste4 and Ras1 interact with the regulatory domain of Byr2 directly. Here, we define the domains of Byr2 that bind Ste4 and Ras1 and show that the Byr2 regulatory domain binds to the catalytic domain in the two-hybrid system. Using Byr2 mutants, we demonstrate that these direct physical interactions are all required for proper signaling. In particular, the physical association between Byr2 regulatory and catalytic domains appears to result in autoinhibition, the loss of which results in kinase activation. Furthermore, we provide evidence that Shk1, the S. pombe homolog of the STE20 protein kinase, can directly antagonize the Byr2 intramolecular interaction, possibly by phosphorylating Byr2.

scholarly journals Dual phosphorylation and autophosphorylation in mitogen-activated protein (MAP) kinase activation

1993 ◽  
Vol 296 (1) ◽  
pp. 25-31 ◽  
Author(s):  
J H Her ◽  
S Lakhani ◽  
K Zu ◽  
J Vila ◽  
P Dent ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Map Kinase ◽  
Specific Protein ◽  
Enzymic Activity ◽  
Extracellular Signal ◽  
Protein Map ◽  
Mitogen Activated Protein ◽  
Catalytic Loop

p42mapk [mitogen activated protein (MAP) kinase; extracellular signal-regulated protein kinase (ERK)] is a serine/threonine-specific protein kinase that is activated by dual tyrosine and threonine phosphorylation in response to diverse agonists. Both the tyrosine and threonine phosphorylations are necessary for full enzymic activity. A MAP kinase activator recently purified and cloned has been shown to be a protein kinase (MAP kinase kinase) that is able to induce the dual phosphorylation of MAP kinase on both the regulatory tyrosine and threonine sites in vitro. In the present paper we have utilized MAP kinase mutants altered in the sites of regulatory phosphorylation to show, both in vivo and in vitro, that phosphorylation of the tyrosine and the threonine can occur independently of one another, with no required order of phosphorylation. We also utilized kinase-defective variants of MAP kinase with mutations in either the ATP-binding loop or the catalytic loop, and obtained data suggesting that the activity or structure of the catalytic loop of MAP kinase plays an important role in its own dual phosphorylation.

scholarly journals Cyclic AMP Selectively Uncouples Mitogen-Activated Protein Kinase Cascades from Activating Signals

2006 ◽  
Vol 26 (8) ◽  
pp. 3039-3047 ◽  
Author(s):  
Gray W. Pearson ◽  
Svetlana Earnest ◽  
Melanie H. Cobb
Keyword(s):  
Growth Factors ◽  
Growth Factor ◽  
Protein Kinase ◽  
Cyclic Amp ◽  
Map Kinase ◽  
Signaling Pathways ◽  
Cellular Response ◽  
Mitogen Activated Protein Kinase ◽  
Extracellular Signal ◽  
Mitogen Activated Protein

ABSTRACT Cells integrate signals to select the appropriate response from an array of possible outcomes. Signal integration causes the reorganization of signaling pathways by undescribed events. To analyze the molecular changes in signaling pathways that elicit different responses, we focused on the interaction between cyclic AMP (cAMP) and growth factors. We show that the activation of extracellular signal-regulated kinase 5 (ERK5), but not ERK1/2, by growth factors is disrupted by cAMP through cAMP-dependent protein kinase (PKA). Activation of MEKK2, a mitogen-activated protein (MAP) kinase kinase kinase upstream of ERK5 that is required for growth factor activation of ERK5, is also disrupted by PKA. Transcription of c-Jun is induced by ERK5, and like ERK5, c-Jun induction is also blocked by cAMP. Transcription from the serum response element, like activation of ERK1/2, is not blocked by cAMP. Collectively, these results support a model in which cAMP shapes the growth factor-induced cellular response through PKA-dependent uncoupling of selected MAP kinase cascades from activating signals.

scholarly journals Sequential Activation of the MEK-Extracellular Signal-Regulated Kinase and MKK3/6-p38 Mitogen-Activated Protein Kinase Pathways Mediates Oncogenic ras-Induced Premature Senescence

2002 ◽  
Vol 22 (10) ◽  
pp. 3389-3403 ◽  
Author(s):  
Weiping Wang ◽  
Joan X. Chen ◽  
Rong Liao ◽  
Qingdong Deng ◽  
Jennifer J. Zhou ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Mammalian Cells ◽  
Human Fibroblasts ◽  
Mitogen Activated Protein Kinase ◽  
Premature Senescence ◽  
Erk Pathway ◽  
Primary Cells ◽  
Extracellular Signal Regulated Kinase ◽  
Extracellular Signal ◽  
Mitogen Activated Protein

ABSTRACT In primary mammalian cells, oncogenic ras induces premature senescence, depending on an active MEK-extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathway. It has been unclear how activation of the mitogenic MEK-ERK pathway by ras can confer growth inhibition. In this study, we have found that the stress-activated MAPK, p38, is also activated during the onset of ras-induced senescence in primary human fibroblasts. Constitutive activation of p38 by active MKK3 or MKK6 induces senescence. Oncogenic ras fails to provoke senescence when p38 activity is inhibited, suggesting that p38 activation is essential for ras-induced senescence. Furthermore, we have demonstrated that p38 activity is stimulated by ras as a result of an activated MEK-ERK pathway. Following activation of MEK and ERK, expression of oncogenic ras leads to the accumulation of active MKK3/6 and p38 activation in a MEK-dependent fashion and subsequently induces senescence. Active MEK1 induces the same set of changes and provokes senescence relying on active p38. Therefore, oncogenic ras provokes premature senescence by sequentially activating the MEK-ERK and MKK3/6-p38 pathways in normal, primary cells. These studies have defined the molecular events within the ras signaling cascade that lead to premature senescence and, thus, have provided new insights into how ras confers oncogenic transformation in primary cells.

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