Direct effect of protein kinase A on four putative phosphorylation sites of aquaporin 2 in vitro

ABSTRACT Sox9 is a high-mobility-group domain-containing transcription factor required for chondrocyte differentiation and cartilage formation. We used a yeast two-hybrid method based on Son of Sevenless (SOS) recruitment to screen a chondrocyte cDNA library and found that the catalytic subunit of cyclic AMP (cAMP)-dependent protein kinase A (PKA-Cα) interacted specifically with SOX9. Next we found that two consensus PKA phosphorylation sites within SOX9 could be phosphorylated by PKA in vitro and that SOX9 could be phosphorylated by PKA-Cα in vivo. In COS-7 cells cotransfected with PKA-Cα and SOX9 expression plasmids, PKA enhanced the phosphorylation of wild-type SOX9 but did not affect phosphorylation of a SOX9 protein in which the two PKA phosphorylation sites (S64 and S211) were mutated. Using a phosphospecific antibody that specifically recognized SOX9 phosphorylated at serine 211, one of the two PKA phosphorylation sites, we demonstrated that addition of cAMP to chondrocytes strongly increased the phosphorylation of endogenous Sox9. In addition, immunohistochemistry of mouse embryo hind legs showed that Sox9 phosphorylated at serine 211 was principally localized in the prehypertrophic zone of the growth plate, corresponding to the major site of expression of the parathyroid hormone-related peptide (PTHrP) receptor. Since cAMP has previously been shown to effectively increase the mRNA levels of Col2a1 and other specific markers of chondrocyte differentiation in culture, we then asked whether PKA phosphorylation could modulate the activity of SOX9. Addition of 8-bromo-cAMP to chondrocytes in culture increased the activity of a transiently transfected SOX9-dependent 48-bp Col2a1chondrocyte-specific enhancer; similarly, cotransfection of PKA-Cα increased the activity of this enhancer. Mutations of the two PKA phosphorylation consensus sites of SOX9 markedly decreased the PKA-Cα activation of this enhancer by SOX9. PKA phosphorylation and the mutations in the consensus PKA phosphorylation sites of SOX9 did not alter its nuclear localization. In vitro phosphorylation of SOX9 by PKA resulted in more efficient DNA binding. We conclude that SOX9 is a target of cAMP signaling and that phosphorylation of SOX9 by PKA enhances its transcriptional and DNA-binding activity. Because PTHrP signaling is mediated by cAMP, our results support the hypothesis that Sox9 is a target of PTHrP signaling in the growth plate and that the increased activity of Sox9 might mediate the effect of PTHrP in maintaining the cells as nonhypertrophic chondrocytes.

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Protein kinase A phosphorylation is involved in regulated exocytosis of aquaporin-2 in transfected LLC-PK1 cells

AJP Renal Physiology ◽

10.1152/ajprenal.1997.272.6.f816 ◽

1997 ◽

Vol 272 (6) ◽

pp. F816-F822 ◽

Cited By ~ 92

Author(s):

T. Katsura ◽

C. E. Gustafson ◽

D. A. Ausiello ◽

D. Brown

Keyword(s):

Plasma Membrane ◽

Protein Kinase ◽

Protein Kinase A ◽

Phosphorylation Site ◽

Transfected Cells ◽

Aquaporin 2 ◽

Intracellular Vesicles ◽

Kinase A

Vasopressin-dependent translocation of aquaporin-2 (AQP2) between intracellular vesicles and the plasma membrane has been demonstrated in vivo and in vitro. Furthermore, the vasopressin-induced increase in apical membrane water permeability of renal principal cells is dependent on a rise in intracellular adenosine 3',5'-cyclic monophosphate and activation of protein kinase A (PKA). To determine whether trafficking of AQP2 is dependent on PKA phosphorylation, we first examined the effect of the PKA-inhibitor N-(2[[3-(4-bromophenyl)-2-propenyl]-amino]-ethyl)-5-isoquinolinesulfonam ide (H-89) on AQP2 translocation in transfected LLC-PK1 cells. Vasopressin-induced membrane insertion of AQP2 was completely inhibited by pretreatment of the cells for 60 min with H-89. This reagent also caused a dense accumulation of AQP2 in the Golgi region. Next, LLC-PK1 cells were stably transfected with AQP2 cDNA in which the PKA phosphorylation site, Ser256, was replaced with alanine (S256A). S256A-AQP2 was not phosphorylated in vitro by PKA, and S256A-AQP2 was mainly localized to intracellular vesicles in the basal condition, similar to wild-type AQP2. However, after stimulation with vasopressin or forskolin, the cellular distribution of S256A-AQP2 remained unchanged. In addition, the usual vasopressin-induced increase in endocytosis seen in AQP2-transfected cells was not observed in S256A-AQP2-transfected cells. These results demonstrate that the Ser256 PKA phosphorylation site is possibly involved in the vasopressin-induced trafficking of AQP2 from intracellular vesicles to the plasma membrane and in the subsequent stimulation of endocytosis.

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Faculty Opinions recommendation of Reciprocal regulation of aquaporin-2 abundance and degradation by protein kinase A and p38-MAP kinase.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.4989960.4921056 ◽

2010 ◽

Author(s):

Bellamkonda Kishore

Keyword(s):

Protein Kinase ◽

Map Kinase ◽

Protein Kinase A ◽

P38 Map Kinase ◽

Reciprocal Regulation ◽

Aquaporin 2 ◽

Kinase A

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In vitro phosphorylation of human complement factor C3 by protein kinase A and protein kinase C. Effects on the classical and alternative pathways.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)39892-8 ◽

1990 ◽

Vol 265 (5) ◽

pp. 2941-2946

Author(s):

P O Forsberg ◽

S C Martin ◽

B Nilsson ◽

P Ekman ◽

U R Nilsson ◽

...

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Protein Kinase A ◽

Complement Factor ◽

Human Complement ◽

Alternative Pathways ◽

Kinase C ◽

Complement Factor C3 ◽

Kinase A

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Identification of the major phosphorylation domain of murine mdr1b P-glycoprotein. Analysis of the protein kinase A and protein kinase C phosphorylation sites.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)74570-0 ◽

1993 ◽

Vol 268 (33) ◽

pp. 25054-25062

Author(s):

G A Orr ◽

E K Han ◽

P C Browne ◽

E Nieves ◽

B M O'Connor ◽

...

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Protein Kinase A ◽

Phosphorylation Sites ◽

Kinase C ◽

P Glycoprotein ◽

Phosphorylation Domain ◽

Kinase A

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Frequency- and Afterload-Dependent Cardiac Modulation In Vivo by Troponin I With Constitutively Active Protein Kinase A Phosphorylation Sites

Circulation Research ◽

10.1161/01.res.0000117307.57798.f5 ◽

2004 ◽

Vol 94 (4) ◽

pp. 496-504 ◽

Cited By ~ 113

Author(s):

Eiki Takimoto ◽

David G. Soergel ◽

Paul M.L. Janssen ◽

Linda B. Stull ◽

David A. Kass ◽

...

Keyword(s):

Protein Kinase ◽

Protein Kinase A ◽

Troponin I ◽

Phosphorylation Sites ◽

Active Protein ◽

Cardiac Modulation ◽

Kinase A ◽

Constitutively Active

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Activity, expression and function of a second Drosophila protein kinase A catalytic subunit gene.

Genetics ◽

10.1093/genetics/141.4.1507 ◽

1995 ◽

Vol 141 (4) ◽

pp. 1507-1520 ◽

Cited By ~ 5

Author(s):

A Meléndez ◽

W Li ◽

D Kalderon

Keyword(s):

Protein Kinase ◽

Protein Kinase A ◽

Essential Gene ◽

Sequence Similarity ◽

Catalytic Subunit ◽

Subunit Gene ◽

Drosophila Protein ◽

Pka Catalytic Subunit ◽

Kinase A

Abstract The DC2 gene was isolated previously on the basis of sequence similarity to DC0, the major Drosophila protein kinase A (PKA) catalytic subunit gene. We show here that the 67-kD Drosophila DC2 protein behaves as a PKA catalytic subunit in vitro. DC2 is transcribed in mesodermal anlagen of early embryos. This expression depends on dorsal but on neither twist nor snail activity. DC2 transcriptional fusions mimic this embryonic expression and are also expressed in subsets of cells in the optic lamina, wing disc and leg discs of third instar larvae. A saturation screen of a small deficiency interval containing DC2 for recessive lethal mutations yielded no DC2 alleles. We therefore isolated new deficiencies to generate deficiency trans-heterozygotes that lacked DC2 activity. These animals were viable and fertile. The absence of DC2 did not affect the viability or phenotype of imaginal disc cells lacking DC0 activity or embryonic hatching of animals with reduced DC0 activity. Furthermore, transgenes expressing DC2 from a DC0 promoter did not efficiently rescue a variety of DC0 mutant phenotypes. These observations indicate that DC2 is not an essential gene and is unlikely to be functionally redundant with DC0, which has multiple unique functions during development.

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