scholarly journals cAMP-dependent protein kinase (PKA) complexes probed by complementary differential scanning fluorimetry and ion mobility–mass spectrometry

2016 ◽  
Vol 473 (19) ◽  
pp. 3159-3175 ◽  
Author(s):  
Dominic P. Byrne ◽  
Matthias Vonderach ◽  
Samantha Ferries ◽  
Philip J. Brownridge ◽  
Claire E. Eyers ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Protein Kinase ◽  
Ion Mobility ◽  
Catalytic Domain ◽  
Ion Mobility Mass Spectrometry ◽  
Dependent Protein Kinase ◽  
Camp Dependent Protein Kinase ◽  
Differential Scanning Fluorimetry ◽  
Dependent Protein

cAMP-dependent protein kinase (PKA) is an archetypal biological signaling module and a model for understanding the regulation of protein kinases. In the present study, we combine biochemistry with differential scanning fluorimetry (DSF) and ion mobility–mass spectrometry (IM–MS) to evaluate effects of phosphorylation and structure on the ligand binding, dynamics and stability of components of heteromeric PKA protein complexes in vitro. We uncover dynamic, conformationally distinct populations of the PKA catalytic subunit with distinct structural stability and susceptibility to the physiological protein inhibitor PKI. Native MS of reconstituted PKA R2C2 holoenzymes reveals variable subunit stoichiometry and holoenzyme ablation by PKI binding. Finally, we find that although a ‘kinase-dead’ PKA catalytic domain cannot bind to ATP in solution, it interacts with several prominent chemical kinase inhibitors. These data demonstrate the combined power of IM–MS and DSF to probe PKA dynamics and regulation, techniques that can be employed to evaluate other protein-ligand complexes, with broad implications for cellular signaling.

scholarly journals Regulation of Microtubule Dynamics by Extracellular Signals: cAMP-dependent Protein Kinase Switches Off the Activity of Oncoprotein 18 in Intact Cells

1998 ◽  
Vol 140 (1) ◽  
pp. 131-141 ◽  
Author(s):  
Helena Melander Gradin ◽  
Niklas Larsson ◽  
Ulrica Marklund ◽  
Martin Gullberg
Keyword(s):  
Protein Kinase ◽  
Genetic System ◽  
Cell Surface Receptor ◽  
In Vitro Assays ◽  
Dependent Protein Kinase ◽  
Intact Cells ◽  
Camp Dependent Protein Kinase ◽  
Oncoprotein 18 ◽  
Dependent Protein

Oncoprotein 18 (Op18, also termed p19, 19K, metablastin, stathmin, and prosolin) is a recently identified regulator of microtubule (MT) dynamics. Op18 is a target for both cell cycle and cell surface receptor-coupled kinase systems, and phosphorylation of Op18 on specific combinations of sites has been shown to switch off its MT-destabilizing activity. Here we show that induced expression of the catalytic subunit of cAMP-dependent protein kinase (PKA) results in a dramatic increase in cellular MT polymer content concomitant with phosphorylation and partial degradation of Op18. That PKA may regulate the MT system by downregulation of Op18 activity was evaluated by a genetic system allowing conditional co-expression of PKA and a series of kinase target site–deficient mutants of Op18. The results show that phosphorylation of Op18 on two specific sites, Ser-16 and Ser-63, is necessary and sufficient for PKA to switch off Op18 activity in intact cells. The regulatory importance of dual phosphorylation on Ser-16 and Ser-63 of Op18 was reproduced by in vitro assays. These results suggest a simple model where PKA phosphorylation downregulates the MT-destabilizing activity of Op18, which in turn promotes increased tubulin polymerization. Hence, the present study shows that Op18 has the potential to regulate the MT system in response to external signals such as cAMP-linked agonists.

scholarly journals Adrenocorticotrophic hormone stimulates phosphotyrosine phosphatase SHP2 in bovine adrenocortical cells: phosphorylation and activation by cAMP-dependent protein kinase

2000 ◽  
Vol 352 (2) ◽  
pp. 483-490 ◽  
Author(s):  
Stéphane ROCCHI ◽  
Isabelle GAILLARD ◽  
Emmanuel VAN OBBERGHEN ◽  
Edmond M. CHAMBAZ ◽  
Isabelle VILGRAIN
Keyword(s):  
Protein Kinase ◽  
Kinase Assay ◽  
Dependent Manner ◽  
Adrenocorticotrophic Hormone ◽  
Adrenocortical Cells ◽  
Dependent Protein Kinase ◽  
Phosphotyrosine Phosphatase ◽  
Camp Dependent Protein Kinase ◽  
Dependent Protein

During activation of adrenocortical cells by adrenocorticotrophic hormone (ACTH), tyrosine dephosphorylation of paxillin is stimulated and this correlates with protrusion of filopodial structures and a decreased number of focal adhesions. These effects are inhibited by Na3VO4, a phosphotyrosine phosphatase inhibitor [Vilgrain, Chinn, Gaillard, Chambaz and Feige (1998) Biochem. J. 332, 533–540]. However, the tyrosine phosphatases involved in these processes remain to be identified. In this study, we provide evidence that the Src homology domain (SH)2-containing phosphotyrosine phosphatase (SHP)2, but not SHP1, is expressed in adrenocortical cells and is phosphorylated upon ACTH challenge. ACTH (10-8M) treatment of 32P-labelled adrenocortical cells resulted in an increase in phosphorylated SHP2. By probing SHP2-containing immunoprecipitates with an antibody to phosphoserine we found that SHP2 was phosphorylated on serine in ACTH-treated cells in a dose- and time-dependent manner. Furthermore, using an in vitro kinase assay, we showed that SHP2 was a target for cAMP-dependent protein kinase (PKA). Serine was identified as the only target amino acid phosphorylated in SHP2. Phosphorylation of SHP2 by PKA resulted in a dramatic stimulation of phosphatase activity measured either with insulin receptor substrate-1 or with the synthetic peptide [32P]poly(Glu/Tyr) as substrate. In an in-gel assay of SHP2-containing immunoprecipitates, phosphorylated in vitro by PKA or isolated from adrenocortical cells treated with 10nM ACTH, a pronounced activation of SHP2 activity was shown. These observations clearly support the idea that a PKA-mediated signal transduction pathway contributes to SHP2 regulation in adrenocortical cells and point to SHP2 as a possible mediator of the effects of ACTH.

scholarly journals Activation of a Nuclear Cdc2-Related Kinase within a Mitogen-Activated Protein Kinase-Like TDY Motif by Autophosphorylation and Cyclin-Dependent Protein Kinase-Activating Kinase

2005 ◽  
Vol 25 (14) ◽  
pp. 6047-6064 ◽  
Author(s):  
Zheng Fu ◽  
Melanie J. Schroeder ◽  
Jeffrey Shabanowitz ◽  
Philipp Kaldis ◽  
Kasumi Togawa ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Nuclear Localization ◽  
Kinase Activity ◽  
Catalytic Domain ◽  
Mitogen Activated Protein Kinase ◽  
Intestinal Cell ◽  
Dependent Protein Kinase ◽  
Mitogen Activated Protein ◽  
Dependent Protein

ABSTRACT Male germ cell-associated kinase (MAK) and intestinal cell kinase (ICK) are nuclear Cdc2-related kinases with nearly identical N-terminal catalytic domains and more divergent C-terminal noncatalytic domains. The catalytic domain is also related to mitogen-activated protein kinases (MAPKs) and contains a corresponding TDY motif. Nuclear localization of ICK requires subdomain XI and interactions of the conserved Arg-272, but not kinase activity or, surprisingly, any of the noncatalytic domain. Further, nuclear localization of ICK is required for its activation. ICK is activated by dual phosphorylation of the TDY motif. Phosphorylation of Tyr-159 in the TDY motif requires ICK autokinase activity but confers only basal kinase activity. Full activation requires additional phosphorylation of Thr-157 in the TDY motif. Coexpression of ICK with constitutively active MEK1 or MEK5 fails to increase ICK phosphorylation or activity, suggesting that MEKs are not involved. ICK and MAK are related to Ime2p in budding yeast, and cyclin-dependent protein kinase-activating kinase Cak1p has been placed genetically upstream of Ime2p. Recombinant Cak1p phosphorylates Thr-157 in the TDY motif of recombinant ICK and activates its activity in vitro. Coexpression of ICK with wild-type CAK1 but not kinase-inactive CAK1 in cells also increases ICK phosphorylation and activity. Our studies establish ICK as the prototype for a new group of MAPK-like kinases requiring dual phosphorylation at TDY motifs.

cAMP-dependent protein kinase mediates hydrosmotic effect of vasopressin in collecting duct

1992 ◽  
Vol 263 (1) ◽  
pp. C147-C153 ◽  
Author(s):  
H. M. Snyder ◽  
T. D. Noland ◽  
M. D. Breyer
Keyword(s):  
Protein Kinase ◽  
Protein Phosphorylation ◽  
Water Permeability ◽  
Collecting Duct ◽  
Regulatory Subunit ◽  
Dependent Protein Kinase ◽  
Camp Dependent Protein Kinase ◽  
Dependent Protein ◽  
Pka Regulatory Subunit

The role of adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase A (PKA) in mediating the hydrosmotic effect of vasopressin in in vitro microperfused rabbit cortical collecting ducts (CCDs) was examined. We measured PKA substrate phosphorylation and water permeability [hydraulic conductivity (Lp) = 10(-7) cm.atm-1.s-1], stimulated by substituted cAMP analogues selective for a unique cAMP binding site (site A or B) on PKA regulatory subunit (R). Synergy between site A- and site B-selective analogues suggests involvement of PKA, because both sites must be occupied for R to dissociate from the catalytic subunit (C), allowing phosphorylation to proceed. As single agents, the site B-selective analogues 8-(4-chlorophenylthio)-cAMP (8-CPT) and 8-thiomethyl-cAMP (8-SCH3) were at least two orders of magnitude more potent than the site A-selective analogues N6-monobutyryl-cAMP (N6-mono) or N6-benzoyl-cAMP (N6-benz). Combinations of subthreshold concentrations of two site A analogues (N6-mono+N6-benz) or two site B-selective analogues (8-CPT + 8-SCH3) failed to significantly increase protein phosphorylation or water permeability. In contrast, combination of a site A plus site B analogue synergistically stimulated both protein phosphorylation and Lp. Rp-cAMPS, an inhibitor of cAMP binding to PKA, reduced both vasopressin (41% inhibition)- and cAMP (56% inhibition)-stimulated water permeability. H-89 (50 microM), an inhibitor of PKA kinase activity, also blocked cAMP-stimulated water permeability (90% inhibition). These findings suggest that vasopressin-induced water permeability in the rabbit CCD is mediated by PKA.

scholarly journals Activation of serum- and glucocorticoid-regulated protein kinase by agonists that activate phosphatidylinositide 3-kinase is mediated by 3-phosphoinositide-dependent protein kinase-1 (PDK1) and PDK2

1999 ◽  
Vol 339 (2) ◽  
pp. 319-328 ◽  
Author(s):  
Takayasu KOBAYASHI ◽  
Philip COHEN
Keyword(s):  
Hydrogen Peroxide ◽  
Protein Kinase ◽  
Catalytic Domain ◽  
Glycogen Synthase ◽  
Pleckstrin Homology Domain ◽  
Dependent Protein Kinase ◽  
293 Cells ◽  
Dependent Protein ◽  
Dependent Pathway

The PtdIns(3,4,5)P3-dependent activation of protein kinase B (PKB) by 3-phosphoinositide-dependent protein kinases-1 and -2 (PDK1 and PDK2 respectively) is a key event in mediating the effects of signals that activate PtdIns 3-kinase. The catalytic domain of serum- and glucocorticoid-regulated protein kinase (SGK) is 54% identical with that of PKB and, although lacking the PtdIns(3,4,5)P3-binding pleckstrin-homology domain, SGK retains the residues that are phosphorylated by PDK1 and PDK2, which are Thr256 and Ser422 in SGK. Here we show that PDK1 activates SGK in vitro by phosphorylating Thr256. We also show that, in response to insulin-like growth factor-1 (IGF-1) or hydrogen peroxide, transfected SGK is activated in 293 cells via a PtdIns 3-kinase-dependent pathway that involves the phosphorylation of Thr256 and Ser422. The activation of SGK by PDK1 in vitro is unaffected by PtdIns(3,4,5)P3, abolished by the mutation of Ser422 to Ala, and greatly potentiated by mutation of Ser422 to Asp (although this mutation does not activate SGK itself). Consistent with these findings, the Ser422Asp mutant of SGK is activated by phosphorylation (probably at Thr256) in unstimulated 293 cells, and activation is unaffected by inhibitors of PtdIns 3-kinase. Our results are consistent with a model in which activation of SGK by IGF-1 or hydrogen peroxide is initiated by a PtdIns(3,4,5)P3-dependent activation of PDK2, which phosphorylates Ser422. This is followed by the PtdIns(3,4,5)P3-independent phosphorylation at Thr256 that activates SGK, and is catalysed by PDK1. Like PKB, SGK preferentially phosphorylates serine and threonine residues that lie in Arg-Xaa-Arg-Xaa-Xaa-Ser/Thr motifs, and SGK and PKB inactivate glycogen synthase kinase-3 similarly in vitroand in co-transfection experiments. These findings raise the possibility that some physiological roles ascribed to PKB on the basis of the overexpression of constitutively active PKB mutants might be mediated by SGK.

Dephosphorylation of cAMP-dependent protein kinase regulatory subunit in stimulated parietal cells

1992 ◽  
Vol 262 (4) ◽  
pp. G763-G773 ◽  
Author(s):  
J. R. Goldenring ◽  
V. A. Asher ◽  
M. F. Barreuther ◽  
J. J. Lewis ◽  
S. M. Lohmann ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Okadaic Acid ◽  
Protein Phosphatase ◽  
Parietal Cells ◽  
Regulatory Subunit ◽  
Dependent Protein Kinase ◽  
Camp Dependent Protein Kinase ◽  
Dependent Protein ◽  
Endogenous Proteins

The phosphorylation of endogenous proteins was investigated in subcellular fractions prepared from isolated rabbit parietal cells incubated with either cimetidine (unstimulated) or a combination of histamine and forskolin (maximally stimulated). Phosphorylation of endogenous proteins in subfractions was then assessed in a post hoc assay using [gamma-32P]ATP as a phosphate donor in vitro. The Mg(2+)-dependent incorporation of [32P]phosphate into a 52-kDa protein (pp52M) was observed in the 4,000 g membrane fraction from stimulated but not unstimulated cells. The pp52M protein was identified as the type II regulatory subunit of adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (RII) by isoelectric focusing, comigration with cAMP-binding proteins, and immunoprecipitation. Incorporation of [32P]phosphate into RII in the in vitro assay in the presence of Zn2+ was apparent in the 4,000 g membrane from stimulated but not unstimulated cells. The results thus suggested that, on stimulation, RII in membrane was dephosphorylated. Incorporation of [32P]phosphate into membrane-associated RII was completely abolished in the presence of 10 microM cAMP. The decrease in RII phosphorylation in membrane from stimulated cells assayed in the presence of cAMP was due to a phosphoprotein phosphatase activity that was completely inhibited by okadaic acid (1 microM). The results indicate that stimulation of parietal cells with histamine and forskolin results in the dephosphorylation of membrane bound RII by a protein phosphatase that is also membrane associated. Furthermore, okadaic acid inhibited histamine-stimulated accumulation of [14C]aminopyrine into isolated parietal cells without altering stimulated increases in cAMP. Thus protein phosphatase may be a significant regulator of parietal cell function.

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