scholarly journals Use of LC-MS/MS and Bayes' theorem to identify protein kinases that phosphorylate aquaporin-2 at Ser256

2014 ◽  
Vol 307 (2) ◽  
pp. C123-C139 ◽  
Author(s):  
Davis Bradford ◽  
Viswanathan Raghuram ◽  
Justin L. L. Wilson ◽  
Chung-Lin Chou ◽  
Jason D. Hoffert ◽  
...  
Keyword(s):  
Experimental Data ◽  
Protein Kinase ◽  
Protein Kinases ◽  
Water Transport ◽  
Collecting Duct ◽  
Bayes Theorem ◽  
Dependent Protein Kinase ◽  
Dependent Protein ◽  
Medullary Collecting Duct

In the renal collecting duct, binding of AVP to the V2 receptor triggers signaling changes that regulate osmotic water transport. Short-term regulation of water transport is dependent on vasopressin-induced phosphorylation of aquaporin-2 (AQP2) at Ser256. The protein kinase that phosphorylates this site is not known. We use Bayes' theorem to rank all 521 rat protein kinases with regard to the likelihood of a role in Ser256 phosphorylation on the basis of prior data and new experimental data. First, prior probabilities were estimated from previous transcriptomic and proteomic profiling data, kinase substrate specificity data, and evidence for kinase regulation by vasopressin. This ranking was updated using new experimental data describing the effects of several small-molecule kinase inhibitors with known inhibitory spectra (H-89, KN-62, KN-93, and GSK-650394) on AQP2 phosphorylation at Ser256 in inner medullary collecting duct suspensions. The top-ranked kinase was Ca2+/calmodulin-dependent protein kinase II (CAMK2), followed by protein kinase A (PKA) and protein kinase B (AKT). Liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based in vitro phosphorylation studies compared the ability of three highly ranked kinases to phosphorylate AQP2 and other inner medullary collecting duct proteins, PKA, CAMK2, and serum/glucocorticoid-regulated kinase (SGK). All three proved capable of phosphorylating AQP2 at Ser256, although CAMK2 and PKA were more potent than SGK. The in vitro phosphorylation experiments also identified candidate protein kinases for several additional phosphoproteins with likely roles in collecting duct regulation, including Nedd4-2, Map4k4, and 3-phosphoinositide-dependent protein kinase 1. We conclude that Bayes' theorem is an effective means of integrating data from multiple data sets in physiology.

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.

In vitro phosphorylation of ciliary dyneins by protein kinases from Paramecium

1993 ◽  
Vol 106 (4) ◽  
pp. 1369-1376 ◽  
Author(s):  
C.E. Walczak ◽  
D.L. Nelson
Keyword(s):  
Protein Kinase ◽  
Protein Kinases ◽  
Sucrose Gradient ◽  
Dependent Protein Kinase ◽  
Cgmp Dependent Protein Kinase ◽  
Camp Dependent Protein Kinase ◽  
Ciliary Protein ◽  
Dependent Protein ◽  
Camp And Cgmp

Paramecium dyneins were tested as substrates for phosphorylation by cAMP-dependent protein kinase, cGMP-dependent protein kinase, and two Ca(2+)-dependent protein kinases that were partially purified from Paramecium extracts. Only cAMP-dependent protein kinase caused significant phosphorylation. The major phosphorylated species was a 29 kDa protein that was present in both 22 S and 12 S dyneins; its phosphate-accepting activity peaked with 22 S dynein. In vitro phosphorylation was maximal at five minutes, then decreased. This decrease in phosphorylation was inhibited by the addition of vanadate or NaF. The 29 kDa protein was not phosphorylated by a heterologous cAMP-dependent protein kinase, the bovine catalytic subunit. Phosphorylation of dynein did not change its ATPase activity. In sucrose gradient fractions from the last step of dynein purification, phosphorylation by an endogenous kinase occurred. This phosphorylation could not be attributed to the small amounts of cAMP- and cGMP-dependent protein kinases known to be present, nor was it Ca(2+)-dependent. This previously uncharacterized ciliary protein kinase used casein as an in vitro substrate.

scholarly journals Possible Intracellular Regulators of Female Sexual Maturation

2015 ◽  
pp. 379-386 ◽  
Author(s):  
A. KOLESAROVA ◽  
A. V. SIROTKIN ◽  
M. MELLEN ◽  
S. ROYCHOUDHURY
Keyword(s):  
Protein Kinase ◽  
Protein Kinases ◽  
Granulosa Cells ◽  
Sexual Maturation ◽  
Cyclin B1 ◽  
Nuclear Antigen ◽  
Dependent Protein Kinase ◽  
Significant Difference ◽  
Dependent Protein

Protein kinases, transcription factors and other apoptosis- and proliferation-related proteins can regulate reproduction, but their involvement in sexual maturation remains to be elucidated. The general aim of the in vivo and in vitro experiments with porcine ovarian granulosa cells was to identify possible intracellular regulators of female sexual maturation. For this purpose, proliferation (expression of proliferating cell nuclear antigen – PCNA, mitogen-activated protein kinases – ERK 1,2 related MAPK and cyclin B1), apoptosis (expression of the apoptotic protein Bax and apoptosis regulator Bcl-2 protein), expression of some protein kinases (cAMP dependent protein kinase – PKA, cGMP-dependent protein kinase – PKG, tyrosine kinase – TK) and cAMP responsive element binding protein 1 (CREB-1) was examined in granulosa cells isolated from ovaries of immature and mature gilts. Expression of PCNA, ERK1,2 related MAPK, cyclin B1, Bcl-2, Bax, PKA, CREB-1, TK and PKG in porcine granulosa cells were detected by immunocytochemistry. Sexual maturation was associated with significant increase in the expression of Bcl-2, Bax, PKA, CREB-1 and TK and with decrease in the expression of ERK1,2 related MAPK, cyclin B1 and PKG in granulosa cells. No significant difference in PCNA expression was noted. The present data obtained from in vitro study indicate that sexual maturation in females is influenced by puberty-related changes in porcine ovarian signaling substances: increase in Bcl-2, Bax, PKA, CREB-1, TK and decrease in ERK1,2 related MAPK, cyclin B1 and PKG. It suggests that these signaling molecules could be potential regulators of porcine sexual maturation.

Molecular mechanisms of ANP inhibition of renal sodium transport

1991 ◽  
Vol 69 (10) ◽  
pp. 1546-1552 ◽  
Author(s):  
Bruce A. Stanton
Keyword(s):  
Protein Kinase ◽  
Cyclic Gmp ◽  
Collecting Duct ◽  
Cation Channel ◽  
Sodium Reabsorption ◽  
Open Probability ◽  
Dependent Protein Kinase ◽  
Inner Medullary Collecting Duct ◽  
Dependent Protein ◽  
Medullary Collecting Duct

ANP, a hormone secreted by the atria of mammalian hearts in response to volume expansion, increases urinary sodium excretion in part by inhibiting sodium reabsorption across the inner medullary collecting duct. A number of nephron segments may contribute to the ANP-induced natriuresis; however, this review will focus on the cellular mechanisms of ANP inhibition of electrogenic sodium reabsorption by the inner medullary collecting duct. Patch-clamp studies conducted on rat inner medullary collecting duct cells in primary culture revealed that ANP, via its second messenger cGMP, inhibits electrogenic sodium reabsorption by reducing the open probability of a cation channel located in the apical membrane. Cyclic GMP inhibits the cation channel and thereby sodium reabsorption by two mechanisms. First, cGMP inhibits the channel by a phosporylation-independent mechanism, by binding either to an allosteric modifier site on the channel or to a regulatory subunit. Second, cGMP inhibits the channel by activating cGMP-dependent protein kinase, which by a sequential pathway involving the GTP-binding protein, Gi inhibits the channel. These cGMP-dependent mechanisms inhibiting sodium reabsorption across the inner medullary collecting duct account for a substantial component of the natriuresis following a rise in ANP levels.Key words: inner medullary collecting duct, G proteins, cyclic GMP-dependent protein kinase, 3′,5′-cyclic GMP, signal transduction, papillary collecting duct.

The activation loop of PKA catalytic isoforms is differentially phosphorylated by Pkh protein kinases in Saccharomyces cerevisiae

2012 ◽  
Vol 448 (3) ◽  
pp. 307-320 ◽  
Author(s):  
Steven Haesendonckx ◽  
Vanesa Tudisca ◽  
Karin Voordeckers ◽  
Silvia Moreno ◽  
Johan M. Thevelein ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Kinase ◽  
Protein Kinases ◽  
Activation Process ◽  
Activation Loop ◽  
Dependent Protein Kinase ◽  
Catalytic Subunits ◽  
Dependent Protein

PDK1 (phosphoinositide-dependent protein kinase 1) phosphorylates and activates PKA (cAMP-dependent protein kinase) in vitro. Docking of the HM (hydrophobic motif) in the C-terminal tail of the PKA catalytic subunits on to the PIF (PDK1-interacting fragment) pocket of PDK1 is a critical step in this activation process. However, PDK1 regulation of PKA in vivo remains controversial. Saccharomyces cerevisiae contains three PKA catalytic subunits, TPK1, TPK2 and TPK3. We demonstrate that Pkh [PKB (protein kinase B)-activating kinase homologue] protein kinases phosphorylate the activation loop of each Tpk in vivo with various efficiencies. Pkh inactivation reduces the interaction of each catalytic subunit with the regulatory subunit Bcy1 without affecting the specific kinase activity of PKA. Comparative analysis of the in vitro interaction and phosphorylation of Tpks by Pkh1 shows that Tpk1 and Tpk2 interact with Pkh1 through an HM–PIF pocket interaction. Unlike Tpk1, mutagenesis of the activation loop site in Tpk2 does not abolish in vitro phosphorylation, suggesting that Tpk2 contains other, as yet uncharacterized, Pkh1 target sites. Tpk3 is poorly phosphorylated on its activation loop site, and this is due to the weak interaction of Tpk3 with Pkh1 because of the atypical HM found in Tpk3. In conclusion, the results of the present study show that Pkh protein kinases contribute to the divergent regulation of the Tpk catalytic subunits.

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