scholarly journals The kinase DYRK1A phosphorylates the transcription factor FKHR at Ser329 in vitro, a novel in vivo phosphorylation site

2001 ◽  
Vol 355 (3) ◽  
pp. 597-607 ◽  
Author(s):  
Yvonne L. WOODS ◽  
Graham RENA ◽  
Nick MORRICE ◽  
Andreas BARTHEL ◽  
Walter BECKER ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Protein Kinase ◽  
Phosphorylation Site ◽  
Control Of Gene Expression ◽  
Cell Extracts ◽  
Dual Specificity ◽  
Dependent Protein ◽  
Phosphoinositide 3 Kinase

Forkhead in rhabdomyosarcoma (FKHR) is a transcription factor that has been implicated in the control of gene expression by insulin, as well as the regulation of apoptosis by survival factors. These signals trigger the protein kinase B (PKB)-catalysed phosphorylation of FKHR at three residues (Thr24, Ser256 and Ser319) by a phosphoinositide 3-kinase-dependent pathway that results in the nuclear exit and inactivation of this transcription factor. Here, we have identified a conserved residue (Ser329) as a novel in vivo phosphorylation site on FKHR. Ser329 phosphorylation also decreases the ability of FKHR to stimulate gene transactivation and reduces the proportion of FKHR present in the nucleus. However, unlike the residues targetted by PKB, Ser329 is phosphorylated in unstimulated HEK-293cells, and phosphorylation is not increased by stimulation with insulin-like growth factor-1 or by transfection with 3-phosphoinositide-dependent protein kinase-1. We have also purified a protein kinase to near homogeneity from rabbit skeletal muscle that phosphorylates FKHR at Ser329 specifically and identified it as DYRK1A (dual-specificity tyrosine-phosphorylated and regulated kinase 1A). We find that FKHR and DYRK1A co-localize in discrete regions of the nucleus and can be co-immunoprecipitated from cell extracts. These experiments suggest that DYRK1A may phosphorylate FKHR at Ser329in vivo.

scholarly journals Mitogen-activated protein kinase kinase 1 (MKK1) is negatively regulated by threonine phosphorylation

1994 ◽  
Vol 14 (3) ◽  
pp. 1594-1602
Author(s):  
A J Rossomando ◽  
P Dent ◽  
T W Sturgill ◽  
D R Marshak
Keyword(s):  
Protein Kinase ◽  
Phosphorylation Site ◽  
Mitogen Activated Protein Kinase ◽  
Dual Specificity ◽  
Mitogen Activated Protein ◽  
Kinase Cascade ◽  
Protein Kinase Cascade ◽  
Protein Kinase Kinase

Mitogen-activated protein kinase kinase 1 (MKK1), a dual-specificity tyrosine/threonine protein kinase, has been shown to be phosphorylated and activated by the raf oncogene product as part of the mitogen-activated protein kinase cascade. Here we report the phosphorylation and inactivation of MKK1 by phosphorylation on threonine 286 and threonine 292. MKK1 contains a consensus phosphorylation site for p34cdc2, a serine/threonine protein kinase that regulates the cell division cycle, at Thr-286 and a related site at Thr-292. p34cdc2 catalyzes the in vitro phosphorylation of MKK1 on both of these threonine residues and inactivates MKK1 enzymatic activity. Both sites are phosphorylated in vivo as well. The data presented in this report provide evidence that MKK1 is negatively regulated by threonine phosphorylation.

scholarly journals Phosphodiesterase 3A binds to 14-3-3 proteins in response to PMA-induced phosphorylation of Ser428

2005 ◽  
Vol 392 (1) ◽  
pp. 163-172 ◽  
Author(s):  
Mercedes Pozuelo Rubio ◽  
David G. Campbell ◽  
Nicholas A. Morrice ◽  
Carol Mackintosh
Keyword(s):  
Protein Kinase ◽  
Metal Ion ◽  
Mitogen Activated Protein Kinase ◽  
Dependent Manner ◽  
Cell Extracts ◽  
Mitogen Activated Protein ◽  
Dependent Protein ◽  
Pkc Protein Kinase C

PDE3A (phosphodiesterase 3A) was identified as a phosphoprotein that co-immunoprecipitates with endogenous 14-3-3 proteins from HeLa cell extracts, and binds directly to 14-3-3 proteins in a phosphorylation-dependent manner. Among cellular stimuli tested, PMA promoted maximal binding of PDE3A to 14-3-3 proteins. While p42/p44 MAPK (mitogen-activated protein kinase), SAPK2 (stress-activated protein kinase 2)/p38 and PKC (protein kinase C) were all activated by PMA in HeLa cells, the PMA-induced binding of PDE3A to 14-3-3 proteins was inhibited by the non-specific PKC inhibitors Ro 318220 and H-7, but not by PD 184352, which inhibits MAPK activation, nor by SB 203580 and BIRB0796, which inhibit SAPK2 activation. Binding of PDE3A to 14-3-3 proteins was also blocked by the DNA replication inhibitors aphidicolin and mimosine, but the PDE3A–14-3-3 interaction was not cell-cycle-regulated. PDE3A isolated from cells was able to bind to 14-3-3 proteins after in vitro phosphorylation with PKC isoforms. Using MS/MS of IMAC (immobilized metal ion affinity chromatography)-enriched tryptic phosphopeptides and phosphospecific antibodies, at least five sites on PDE3A were found to be phosphorylated in vivo, of which Ser428 was selectively phosphorylated in response to PMA and dephosphorylated in cells treated with aphidicolin and mimosine. Phosphorylation of Ser428 therefore correlated with 14-3-3 binding to PDE3A. Ser312 of PDE3A was phosphorylated in an H-89-sensitive response to forskolin, indicative of phosphorylation by PKA (cAMP-dependent protein kinase), but phosphorylation at this site did not stimulate 14-3-3 binding. Thus 14-3-3 proteins can discriminate between sites in a region of multisite phosphorylation on PDE3A. An additional observation was that the cytoskeletal cross-linker protein plectin-1 coimmunoprecipitated with PDE3A independently of 14-3-3 binding.

scholarly journals Alteration of a cyclic AMP-dependent protein kinase phosphorylation site in the c-Fos protein augments its transforming potential

1992 ◽  
Vol 12 (3) ◽  
pp. 998-1006
Author(s):  
I Tratner ◽  
R Ofir ◽  
I M Verma
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Phosphorylation Site ◽  
Dependent Protein Kinase ◽  
Fos Protein ◽  
Dependent Protein ◽  
Cell Growth And Differentiation ◽  
Kinase Phosphorylation

We have studied the phosphorylation of the nuclear oncoprotein Fos by cyclic AMP-dependent protein kinase (PKA). We demonstrate that the human c-Fos protein, phosphorylated either in vitro with purified PKA or in vivo in JEG3 cells following treatment with forskolin, has similar phosphotryptic peptide maps. Serine 362, which constitutes part of a canonical PKA phosphorylation site (RKGSSS), is phosphorylated both in vivo and in vitro. A mutant of Fos protein in which serine residues 362 to 364 have been altered to alanine residues is not efficiently phosphorylated in vitro. Furthermore, Fos protein in which serines 362 to 364 have been altered to alanine shows increased transforming potential. We propose that phosphorylation of Fos by PKA is an important regulatory step in controlling its activity in normal cell growth and differentiation.

scholarly journals Phosphorylation of CK1δ: identification of Ser370 as the major phosphorylation site targeted by PKA in vitro and in vivo

2007 ◽  
Vol 406 (3) ◽  
pp. 389-398 ◽  
Author(s):  
Georgios Giamas ◽  
Heidrun Hirner ◽  
Levani Shoshiashvili ◽  
Arnhild Grothey ◽  
Susanne Gessert ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Phosphorylation Site ◽  
Fold Increase ◽  
Regulatory Subunit ◽  
Kinetic Properties ◽  
Casein Kinase 1 ◽  
Cell Extracts ◽  
Anchoring Protein

The involvement of CK1 (casein kinase 1) δ in the regulation of multiple cellular processes implies a tight regulation of its activity on many different levels. At the protein level, reversible phosphorylation plays an important role in modulating the activity of CK1δ. In the present study, we show that PKA (cAMP-dependent protein kinase), Akt (protein kinase B), CLK2 (CDC-like kinase 2) and PKC (protein kinase C) α all phosphorylate CK1δ. PKA was identified as the major cellular CK1δCK (CK1δ C-terminal-targeted protein kinase) for the phosphorylation of CK1δ in vitro and in vivo. This was implied by the following evidence: PKA was detectable in the CK1δCK peak fraction of fractionated MiaPaCa-2 cell extracts, PKA shared nearly identical kinetic properties with those of CK1δCK, and both PKA and CK1δCK phosphorylated CK1δ at Ser370in vitro. Furthermore, phosphorylation of CK1δ by PKA decreased substrate phosphorylation of CK1δ in vitro. Mutation of Ser370 to alanine increased the phosphorylation affinity of CK1δ for β-casein and the GST (gluthatione S-transferase)–p53 1–64 fusion protein in vitro and enhanced the formation of an ectopic dorsal axis during Xenopus laevis development. Anchoring of PKA and CK1δ to centrosomes was mediated by AKAP (A-kinase-anchoring protein) 450. Interestingly, pre-incubation of MiaPaCa-2 cells with the synthetic peptide St-Ht31, which prevents binding between AKAP450 and the regulatory subunit RII of PKA, resulted in a 6-fold increase in the activity of CK1δ. In summary, we conclude that PKA phosphorylates CK1δ, predominantly at Ser370in vitro and in vivo, and that site-specific phosphorylation of CK1δ by PKA plays an important role in modulating CK1δ-dependent processes.

scholarly journals Alteration of a cyclic AMP-dependent protein kinase phosphorylation site in the c-Fos protein augments its transforming potential.

1992 ◽  
Vol 12 (3) ◽  
pp. 998-1006 ◽  
Author(s):  
I Tratner ◽  
R Ofir ◽  
I M Verma
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Phosphorylation Site ◽  
Dependent Protein Kinase ◽  
Fos Protein ◽  
Dependent Protein ◽  
Cell Growth And Differentiation ◽  
Kinase Phosphorylation

We have studied the phosphorylation of the nuclear oncoprotein Fos by cyclic AMP-dependent protein kinase (PKA). We demonstrate that the human c-Fos protein, phosphorylated either in vitro with purified PKA or in vivo in JEG3 cells following treatment with forskolin, has similar phosphotryptic peptide maps. Serine 362, which constitutes part of a canonical PKA phosphorylation site (RKGSSS), is phosphorylated both in vivo and in vitro. A mutant of Fos protein in which serine residues 362 to 364 have been altered to alanine residues is not efficiently phosphorylated in vitro. Furthermore, Fos protein in which serines 362 to 364 have been altered to alanine shows increased transforming potential. We propose that phosphorylation of Fos by PKA is an important regulatory step in controlling its activity in normal cell growth and differentiation.

scholarly journals Characterization of the structure and regulation of two novel isoforms of serum- and glucocorticoid-induced protein kinase

1999 ◽  
Vol 344 (1) ◽  
pp. 189-197 ◽  
Author(s):  
Takayasu KOBAYASHI ◽  
Maria DEAK ◽  
Nick MORRICE ◽  
Philip COHEN
Keyword(s):  
Protein Kinase ◽  
Catalytic Domain ◽  
Phosphorylation Site ◽  
H4iie Cells ◽  
Dependent Protein ◽  
Phosphorylation Mechanism ◽  
Novel Isoforms

The catalytic domain of serum- and glucocorticoid-induced protein kinase (SGK) is 54% identical with protein kinase B (PKB) and, like PKB, is activated in vitro by 3-phosphoinositide-dependent protein kinase-1 (PDK1) and in vivo in response to signals that activate phosphatidylinositol (PI) 3-kinase. Here we identify two novel isoforms of SGK, termed SGK2 and SGK3, whose catalytic domains share 80% amino acid sequence identity with each other and with SGK (renamed SGK1). Like SGK1, the mRNA encoding SGK3 is expressed in all tissues examined, but SGK2 mRNA is only present at significant levels in liver, kidney and pancreas and, at lower levels, in the brain. The levels of SGK2 mRNA in H4IIE cells and SGK3 mRNA in Rat2 fibroblasts are not increased by stimulation with serum or dexamethasone, whereas the level of SGK1 mRNA is increased greatly. SGK2 and SGK3 are activated in vitro by PDK1, albeit more slowly than SGK1, and their activation is accompanied by the phosphorylation of Thr193 and Thr253 respectively, the residues equivalent to the Thr in the ‘activation loop’ of PKB that is targeted by PDK1. The PDK1-catalysed phosphorylation and activation of SGK2 and SGK3, like SGK1, is greatly potentiated by mutating Ser356 and Ser419 respectively to Asp, these residues being equivalent to the C-terminal phosphorylation site of PKB. Like SGK1, SGK2 and SGK3 are activated 5-fold via a phosphorylation mechanism when cells are exposed to H2O2 but, in contrast with SGK1, activation is only suppressed partially by inhibitors of PI 3-kinase. SGK2 and SGK3 are activated to a smaller extent by insulin-like growth factor-1 (2-fold) than SGK1 (5-fold). Like PKB and SGK1, SGK2 and SGK3 preferentially phosphorylate Ser and Thr residues that lie in Arg-Xaa-Arg-Xaa-Xaa-Ser/Thr motifs.

scholarly journals Mitogen-activated protein kinase kinase 1 (MKK1) is negatively regulated by threonine phosphorylation.

1994 ◽  
Vol 14 (3) ◽  
pp. 1594-1602 ◽  
Author(s):  
A J Rossomando ◽  
P Dent ◽  
T W Sturgill ◽  
D R Marshak
Keyword(s):  
Protein Kinase ◽  
Phosphorylation Site ◽  
Mitogen Activated Protein Kinase ◽  
Dual Specificity ◽  
Mitogen Activated Protein ◽  
Kinase Cascade ◽  
Protein Kinase Cascade ◽  
Protein Kinase Kinase

Mitogen-activated protein kinase kinase 1 (MKK1), a dual-specificity tyrosine/threonine protein kinase, has been shown to be phosphorylated and activated by the raf oncogene product as part of the mitogen-activated protein kinase cascade. Here we report the phosphorylation and inactivation of MKK1 by phosphorylation on threonine 286 and threonine 292. MKK1 contains a consensus phosphorylation site for p34cdc2, a serine/threonine protein kinase that regulates the cell division cycle, at Thr-286 and a related site at Thr-292. p34cdc2 catalyzes the in vitro phosphorylation of MKK1 on both of these threonine residues and inactivates MKK1 enzymatic activity. Both sites are phosphorylated in vivo as well. The data presented in this report provide evidence that MKK1 is negatively regulated by threonine phosphorylation.

scholarly journals Anti-echinococcal effect of verapamil involving the regulation of the calcium/calmodulin-dependent protein kinase II response in vitro and in a murine infection model

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Hai-Jun Gao ◽  
Xu-Dong Sun ◽  
Yan-Ping Luo ◽  
Hua-Sheng Pang ◽  
Xing-Ming Ma ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Mrna Expression ◽  
Echinococcus Granulosus ◽  
Calcium Content ◽  
Mrna Levels ◽  
Dependent Protein Kinase ◽  
Calcium Calmodulin Dependent ◽  
Dependent Protein

Abstract Background Echinococcosis, which is caused by the larvae of cestodes of the genus Echinococcus, is a parasitic zoonosis that poses a serious threat to the health of humans and animals globally. Albendazole is the drug of choice for the treatment of echinococcosis, but it is difficult to meet clinical goals with this chemotherapy due to its low cure rate and associated side effects after its long-term use. Hence, novel anti-parasitic targets and effective treatment alternatives are urgently needed. A previous study showed that verapamil (Vepm) can suppress the growth of Echinococcus granulosus larvae; however, the mechanism of this effect remains unclear. The aim of the present study was to gain insight into the anti-echinococcal effect of Vepm on Echinococcus with a particular focus on the regulatory effect of Vepm on calcium/calmodulin-dependent protein kinase II (Ca2+/CaM-CaMKII) in infected mice. Methods The anti-echinococcal effects of Vepm on Echinococcus granulosus protoscoleces (PSC) in vitro and Echinococcus multilocularis metacestodes in infected mice were assessed. The morphological alterations in Echinococcus spp. induced by Vepm were observed by scanning electron microscopy (SEM), and the changes in calcium content in both the parasite and mouse serum and liver were measured by SEM-energy dispersive spectrometry, inductively coupled plasma mass spectrometry and alizarin red staining. Additionally, the changes in the protein and mRNA levels of CaM and CaMKII in infected mice, and in the mRNA levels of CaMKII in E. granulosus PSC, were evaluated after treatment with Vepm by immunohistochemistry and/or real-time quantitative polymerase chain reaction. Results In vitro, E. granulosus PSC could be killed by Vepm at a concentration of 0.5 μg/ml or higher within 8 days. Under these conditions, the ultrastructure of PSC was damaged, and this damage was accompanied by obvious calcium loss and downregulation of CaMKII mRNA expression. In vivo, the weight and the calcium content of E. multilocularis metacestodes from mice were reduced after treatment with 40 mg/kg Vepm, and an elevation of the calcium content in the sera and livers of infected mice was observed. In addition, downregulation of CaM and CaMKII protein and mRNA expression in the livers of mice infected with E. multilocularis metacestodes was found after treatment with Vepm. Conclusions Vepm exerted a parasiticidal effect against Echinococcus both in vitro and in vivo through downregulating the expression of Ca2+/CaM-CaMKII, which was over-activated by parasitic infection. The results suggest that Ca2+/CaM-CaMKII may be a novel drug target, and that Vepm is a potential anti-echinococcal drug for the future control of echinococcosis.

ADR1c mutations enhance the ability of ADR1 to activate transcription by a mechanism that is independent of effects on cyclic AMP-dependent protein kinase phosphorylation of Ser-230

1992 ◽  
Vol 12 (4) ◽  
pp. 1507-1514
Author(s):  
C L Denis ◽  
S C Fontaine ◽  
D Chase ◽  
B E Kemp ◽  
L T Bemis
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Transcriptional Activation ◽  
Growth Conditions ◽  
Dependent Protein Kinase ◽  
Inactive Form ◽  
Dependent Protein ◽  
Kinase Phosphorylation

Four ADR1c mutations that occur close to Ser-230 of the Saccharomyces cerevisiae transcriptional activator ADR1 and which greatly enhance the ability of ADR1 to activate ADH2 expression under glucose-repressed conditions have been shown to reduce or eliminate cyclic AMP-dependent protein kinase (cAPK) phosphorylation of Ser-230 in vitro. In addition, unregulated cAPK expression in vivo blocks ADH2 depression in an ADR1-dependent fashion in which ADR1c mutations display decreased sensitivity to unregulated cAPK activity. Taken together, these data have suggested that ADR1c mutations enhance ADR1 activity by blocking cAPK phosphorylation and inactivation of Ser-230. We have isolated and characterized an additional 17 ADR1c mutations, defining 10 different amino acid changes, that were located in the region defined by amino acids 227 through 239 of ADR1. Three observations, however, indicate that the ADR1c phenotype is not simply equivalent to a lack of cAPK phosphorylation. First, only some of these newly isolated ADR1c mutations affected the ability of yeast cAPK to phosphorylate corresponding synthetic peptides modeled on the 222 to 234 region of ADR1 in vitro. Second, we observed that strains lacking cAPK activity did not display enhanced ADH2 expression under glucose growth conditions. Third, when Ser-230 was mutated to a nonphosphorylatable residue, lack of cAPK activity led to a substantial increase in ADH2 expression under glucose-repressed conditions. Thus, while cAPK controls ADH2 expression and ADR1 is required for this control, cAPK acts by a mechanism that is independent of effects on ADR1 Ser-230. It was also observed that deletion of the ADR1c region resulted in an ADR1c phenotype. The ADR1c region is, therefore, involved in maintaining ADR1 in an inactive form. ADR1c mutations may block the binding of a repressor to ADR1 or alter the structure of ADR1 so that transcriptional activation regions become unmasked.

scholarly journals Engineering a bioluminescent indicator for cyclic AMP-dependent protein kinase

1991 ◽  
Vol 279 (3) ◽  
pp. 727-732 ◽  
Author(s):  
G B Sala-Newby ◽  
A K Campbell
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Specific Activity ◽  
Phosphorylation Site ◽  
Live Cells ◽  
Phosphorylation Sites ◽  
Dependent Protein Kinase ◽  
Ph Optimum ◽  
Dependent Protein

cDNA coding for the luciferase in the firefly Photinus pyralis was amplified in vitro to generate cyclic AMP-dependent protein kinase phosphorylation sites. The DNA was transcribed and translated to generate light-emitting protein. A valine at position 217 was mutated to arginine to generate a site RRFS and the heptapeptide kemptide, the phosphorylation site of the porcine pyruvate kinase, was added at the N- or C-terminus of the luciferase. The proteins carrying phosphorylation sites were characterized for their specific activity, pI, effect of pH on the colour of the light emitted and effect of the catalytic subunit of protein kinase A in the presence of ATP. Only one of the recombinant proteins (RRFS) was significantly different from wild-type luciferase. The RRFS mutant had a lower specific activity, lower pH optimum, emitted greener light at low pH and when phosphorylated it decreased its activity by up to 80%. This latter effect was reversed by phosphatase. This recombinant protein is a good candidate to measure for the first time cyclic AMP-dependent phosphorylation in live cells.

Sign in / Sign up

Export Citation Format

Share Document