Regulation of Na+-K+-ATPase by cAMP-dependent protein kinase anchored on membrane via its anchoring protein

2000 ◽  
Vol 279 (5) ◽  
pp. C1516-C1527 ◽  
Author(s):  
Kinji Kurihara ◽  
Nobuo Nakanishi ◽  
Takao Ueha
Keyword(s):  
Protein Kinase ◽  
Basolateral Membrane ◽  
Catalytic Subunit ◽  
Regulatory Subunit ◽  
Parotid Glands ◽  
Α Subunit ◽  
Anchoring Protein ◽  
Membrane Bound ◽  
Rat Parotid

Na+-K+- ATPase α-subunits in basolateral membrane vesicles (BLMVs) purified from rat parotid glands were 32P-labeled within 5 s by incubation with [γ-32P]ATP at 37°C in the presence of cAMP, but no labeling occurred without cAMP. Phosphorylation of Na+-K+-ATPase was associated with a decrease in its activity. This α-subunit phosphorylation disappeared when BLMVs were briefly incubated with cAMP and subsequent washing before the incubation with [γ-32P]ATP, indicating that catalytic subunit of protein kinase A (PKA) associated to BLMVs via binding with its RII regulatory subunit anchored on the membrane. In the absence of cAMP, a PKA catalytic subunit readily reassociated with the membrane-bound RII subunit. HT-31 peptide inhibited the Na+-K+-ATPase phosphorylation by membrane-bound endogenous PKA, indicating an involvement of A-kinase anchoring protein (AKAP). AKAP-150 protein in BLMVs was shown by immunoblotting and an RII overlay assay and was coimmunoprecipitated by anti-RII antibody. These results show that Na+-K+-ATPase of rat parotid gland acinar cells is regulated in vivo by membrane-anchored PKA via AKAP rather than by free cytosolic PKA.

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.

Intracellular Elemental Concentrations in Resting and Secreting Rat Parotid Glands

1987 ◽  
Vol 66 (2) ◽  
pp. 537-540 ◽  
Author(s):  
K.T. Izutsu ◽  
D.E. Johnson ◽  
M. Goddard
Keyword(s):  
Secretory Granules ◽  
Dry Weight ◽  
Parotid Glands ◽  
X Ray ◽  
Elemental Concentrations ◽  
Saliva Secretion ◽  
Concentration Changes ◽  
Rat Parotid ◽  
Micro Analysis

Electron probe x-ray micro-analysis was used to study the elemental concentration changes that occur during pilocarpine-stimulated saliva secretion. Quantitative x-ray micro-analysis of elemental concentrations in intracellular compartments of rat parotid glands stimulated in vivo with pilocarpine showed that Na concentration was significantly increased, while K concentration was significantly reduced. The magnitude of these changes was consistent with values obtained in other tissues with the x-ray micro-analysis method, and in the same tissue with other experimental methods. Comparisons with results from studies utilizing dispersed acini suggest that acinar dispersion procedures may affect intracellular elemental concentrations. Total electrolyte concentrations in cytoplasm and secretory granules were estimated to increase on a dry-weight basis following pilocarpine stimulation. The former change is consistent with the notion of a trans-cellular route of salivary fluid flow, while the latter change may be important in the exocytosis of secretory granules.

AQP2 is a substrate for endogenous PP2B activity within an inner medullary AKAP-signaling complex

2001 ◽  
Vol 281 (5) ◽  
pp. F958-F965 ◽  
Author(s):  
Inho Jo ◽  
Donald T. Ward ◽  
Michelle A. Baum ◽  
John D. Scott ◽  
Vincent M. Coghlan ◽  
...  
Keyword(s):  
Hippocampal Neurons ◽  
Collecting Duct ◽  
Rat Kidney ◽  
Regulatory Subunit ◽  
Relative Molecular Mass ◽  
Signaling Complex ◽  
Anchoring Protein ◽  
Pka Regulatory Subunit

We have demonstrated that inner medullary collecting duct (IMCD) heavy endosomes purified from rat kidney IMCD contain the type II protein kinase A (PKA) regulatory subunit (RII), protein phosphatase (PP)2B, PKCζ, and an RII-binding protein (relative molecular mass ∼90 kDa) representing a putative A kinase anchoring protein (AKAP). Affinity chromatography of detergent-solubilized endosomes on cAMP-agarose permits recovery of a protein complex consisting of the 90-kDa AKAP, RII, PP2B, and PKCζ. With the use of small-particle flow cytometry, RII and PKCζ were localized to an identical population of endosomes, suggesting that these proteins are components of an endosomal multiprotein complex.32P-labeled aquaporin-2 (AQP2) present in these PKA-phosphorylated endosomes was dephosphorylated in vitro by either addition of exogenous PP2B or by an endogenous endosomal phosphatase that was inhibited by the PP2B inhibitors EDTA and the cyclophilin-cyclosporin A complex. We conclude that IMCD heavy endosomes possess an AKAP multiprotein-signaling complex similar to that described previously in hippocampal neurons. This signaling complex potentially mediates the phosphorylation of AQP2 to regulate its trafficking into the IMCD apical membrane. In addition, the PP2B component of the AKAP-signaling complex could also dephosphorylate AQP2 in vivo.

scholarly journals PRKAA1 Promotes Proliferation and Inhibits Apoptosis of Gastric Cancer Cells Through Activating JNK1 and Akt Pathways

2020 ◽  
Vol 28 (3) ◽  
pp. 213-223 ◽  
Author(s):  
Yangmei Zhang ◽  
Xichang Zhou ◽  
Long Cheng ◽  
Xiang Wang ◽  
Qinglin Zhang ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Protein Kinase ◽  
Cancer Cells ◽  
Akt Signaling ◽  
Catalytic Subunit ◽  
Gastric Cancer Cells ◽  
In Vivo Experiments ◽  
Compound C ◽  
Amp Activated Protein Kinase

PRKAA1 (protein kinase AMP-activated catalytic subunit α 1) is a catalytic subunit of AMP-activated protein kinase (AMPK), which plays a key role in regulating cellular energy metabolism through phosphorylation, and genetic variations in the PRKAA1 have been found to be associated with gastric cancer risk. However, the effect and underlying molecular mechanism of PRKAA1 on gastric cancer tumorigenesis, especially the proliferation and apoptosis, are not fully understood. Our data showed that PRKAA1 is highly expressed in BGC-823 and MKN45 cells and is expressed low in SGC-7901 and MGC-803 cells in comparison with the other gastric cancer cells. PRKAA1 downregulation by shRNA or treatment of AMPK inhibitor compound C significantly inhibited proliferation as well as promoted cell cycle arrest and apoptosis of BGC-823 and MKN45 cells. Moreover, the expression of PCNA and Bcl-2 and the activity of JNK1 and Akt signaling were also reduced in BGC-823 and MKN45 cells after PRKAA1 downregulation. In vivo experiments demonstrated that tumor growth in nude mice was significantly inhibited after PRKAA1 silencing. Importantly, inactivation of JNK1 or Akt signaling pathway significantly inhibited PRKAA1 overexpression-induced increased cell proliferation and decreased cell apoptosis in MGC-803 cells. In conclusion, our findings suggest that PRKAA1 increases proliferation and restrains apoptosis of gastric cancer cells through activating JNK1 and Akt pathways.

scholarly journals Membrane-Bound Protein Kinase A Inhibits Smooth Muscle Cell Proliferation In Vitro and In Vivo by Amplifying cAMP–Protein Kinase A Signals

2001 ◽  
Vol 88 (3) ◽  
pp. 319-324 ◽  
Author(s):  
Ciro Indolfi ◽  
Eugenio Stabile ◽  
Carmela Coppola ◽  
Adriana Gallo ◽  
Cinzia Perrino ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Smooth Muscle ◽  
Protein Kinase ◽  
Smooth Muscle Cell ◽  
Protein Kinase A ◽  
Membrane Bound ◽  
Proliferation In Vitro ◽  
Kinase A

Hypoxia and AMP independently regulate AMP-activated protein kinase activity in heart

2005 ◽  
Vol 288 (5) ◽  
pp. H2412-H2421 ◽  
Author(s):  
Markus Frederich ◽  
Li Zhang ◽  
James A. Balschi
Keyword(s):  
Protein Kinase ◽  
Metabolic Inhibitors ◽  
Protein Kinase Activity ◽  
Α Subunit ◽  
Rat Hearts ◽  
Upstream Kinase ◽  
Ampk Activity ◽  
Amp Activated Protein Kinase

The hypothesis was tested that hypoxia increases AMP-activated protein kinase (AMPK) activity independently of AMP concentration ([AMP]) in heart. In isolated perfused rat hearts, cytosolic [AMP] was changed from 0.2 to 16 μM using metabolic inhibitors during both normal oxygenation (95% O2-5% CO2, normoxia) and limited oxygenation (95% N2-5% CO2, hypoxia). Total AMPK activity measured in vitro ranged from 2 to 40 pmol·min−1·mg protein−1 in normoxic hearts and from 5 to 55 pmol·min−1·mg protein−1 in hypoxic hearts. The dependence of the in vitro total AMPK activity on the in vivo cytosolic [AMP] was determined by fitting the measurements from individual hearts to a hyperbolic equation. The [AMP] resulting in half-maximal total AMPK activity ( A0.5) was 3 ± 1 μM for hypoxic hearts and 28 ± 13 μM for normoxic hearts. The A0.5 for α2-isoform AMPK activity was 2 ± 1 μM for hypoxic hearts and 13 ± 8 μM for normoxic hearts. Total AMPK activity correlated with the phosphorylation of the Thr172 residue of the AMPK α-subunit. In potassium-arrested hearts perfused with variable O2 content, α-subunit Thr172 phosphorylation increased at O2 ≤ 21% even though [AMP] was <0.3 μM. Thus hypoxia or O2 ≤ 21% increased AMPK phosphorylation and activity independently of cytosolic [AMP]. The hypoxic increase in AMPK activity may result from either direct phosphorylation of Thr172 by an upstream kinase or reduction in the A0.5 for [AMP].

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