patched overexpression alters wing disc size and pattern: transcriptional and post-transcriptional effects on hedgehog targets

Development ◽  
1995 ◽  
Vol 121 (12) ◽  
pp. 4161-4170 ◽  
Author(s):  
R.L. Johnson ◽  
J.K. Grenier ◽  
M.P. Scott
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Target Genes ◽  
Critical Role ◽  
Wing Disc ◽  
Wing Vein ◽  
Anterior Compartment ◽  
Protein Levels ◽  
Anterior Posterior ◽  
Kinase A

The membrane protein, Patched, plays a critical role in patterning embryonic and imaginal tissues in Drosophila. patched constitutively inactivates the transcription of target genes such as wingless, decapentaplegic, and patched itself. The secreted protein, Hedgehog, induces transcription of target genes by opposing the Patched signaling pathway. Using the Gal4 UAS system we have overexpressed patched in wing imaginal discs and found that high Patched levels, expressed in either normal or ectopic patterns, result in loss of wing vein patterning in both compartments centering at the anterior/posterior border. In addition, patched inhibits the formation of the mechanosensory neurons, the campaniform sensilla, in the wing blade. The patched wing vein phenotype is modulated by mutations in hedgehog and cubitus interruptus (ci). Patched overexpression inhibits transcription of patched and decapentaplegic and post-transcriptionally decreases the amount of Ci protein at the anterior/posterior boundary. In hedgehogMrt wing discs, which express ectopic hedgehog, Ci levels are correspondingly elevated, suggesting that hedgehog relieves patched repression of Ci accumulation. Protein kinase A also regulates Ci; protein kinase A mutant clones in the anterior compartment have increased levels of Ci protein. Thus patched influences wing disc patterning by decreasing Ci protein levels and inactivating hedgehog target genes in the anterior compartment.

Proteolysis of cubitus interruptus in Drosophila requires phosphorylation by protein kinase A

Development ◽  
1999 ◽  
Vol 126 (19) ◽  
pp. 4331-4339 ◽  
Author(s):  
M.A. Price ◽  
D. Kalderon
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Hedgehog Signaling ◽  
Target Genes ◽  
Ectopic Expression ◽  
Full Length ◽  
Mutant Form ◽  
Wild Type ◽  
Cubitus Interruptus ◽  
Kinase A

The Hedgehog signal transduction pathway is involved in diverse patterning events in many organisms. In Drosophila, Hedgehog signaling regulates transcription of target genes by modifying the activity of the DNA-binding protein Cubitus interruptus (Ci). Hedgehog signaling inhibits proteolytic cleavage of full-length Ci (Ci-155) to Ci-75, a form that represses some target genes, and also converts the full-length form to a potent transcriptional activator. Reduction of protein kinase A (PKA) activity also leads to accumulation of full-length Ci and to ectopic expression of Hedgehog target genes, prompting the hypothesis that PKA might normally promote cleavage to Ci-75 by directly phosphorylating Ci-155. Here we show that a mutant form of Ci lacking five potential PKA phosphorylation sites (Ci5m) is not detectably cleaved to Ci-75 in Drosophila embryos. Moreover, changes in PKA activity dramatically altered levels of full-length wild-type Ci in embryos and imaginal discs, but did not significantly alter full-length Ci5m levels. We corroborate these results by showing that Ci5m is more active than wild-type Ci at inducing ectopic transcription of the Hh target gene wingless in embryos and that inhibition of PKA enhances induction of wingless by wild-type Ci but not by Ci5m. We therefore propose that PKA phosphorylation of Ci is required for the proteolysis of Ci-155 to Ci-75 in vivo. We also show that the activity of Ci5m remains Hedgehog responsive if expressed at low levels, providing further evidence that the full-length form of Ci undergoes a Hedgehog-dependent activation step.

scholarly journals PRKAR1A gene analysis and protein kinase A activity in endometrial tumors

2012 ◽  
Vol 19 (4) ◽  
pp. 457-462 ◽  
Author(s):  
A Tsigginou ◽  
E Bimpaki ◽  
M Nesterova ◽  
A Horvath ◽  
S Boikos ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Regulatory Subunit ◽  
Normal Endometrium ◽  
Protein Levels ◽  
Normal Tissues ◽  
Subunit Expression ◽  
Dependent Protein ◽  
Pka Regulatory Subunit ◽  
Kinase A

PRKAR1A codes for the type 1a regulatory subunit (RIα) of the cAMP-dependent protein kinase A (PKA), an enzyme with an important role in cell cycle regulation and proliferation. PKA dysregulation has been found in various tumors, and PRKAR1A-inactivating mutations have been reported in mostly endocrine neoplasias. In this study, we investigated PKA activity and the PRKAR1A gene in normal and tumor endometrium. Specimens were collected from 31 patients with endometrial cancer. We used as controls 41 samples of endometrium that were collected from surrounding normal tissues or from women undergoing gynecological operations for other reasons. In all samples, we sequenced the PRKAR1A-coding sequence and studied PKA subunit expression; we also determined PKA activity and cAMP binding. PRKAR1A mutations were not found. However, PKA regulatory subunit protein levels, both RIα and those of regulatory subunit type 2b (RIIβ), were lower in tumor samples; cAMP binding was also lower in tumors compared with normal endometrium (P<0.01). Free PKA activity was higher in tumor samples compared with that of control tissue (P<0.01). There are significant PKA enzymatic abnormalities in tumors of the endometrium compared with surrounding normal tissue; as these were not due to PRKAR1A mutations, other mechanisms affecting PKA function ought to be explored.

scholarly journals Genetic Evidence for a Protein Kinase A/Cubitus Interruptus Complex That Facilitates Processing of Cubitus Interruptus in Drosophila

Genetics ◽  
2001 ◽  
Vol 158 (3) ◽  
pp. 1157-1166
Author(s):  
John A Kiger ◽  
Cristin O'Shea
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Target Gene ◽  
Target Genes ◽  
Signal Transduction Pathway ◽  
Transduction Pathway ◽  
Cubitus Interruptus ◽  
Mutant Forms ◽  
The Relationship ◽  
Kinase A

Abstract Hedgehog (Hh) activates a signal transduction pathway regulating Cubitus interruptus (Ci). In the absence of Hh, full-length Ci (Ci-155) is bound in a complex that includes Costal2 (Cos2) and Fused (Fu). Ci-155 is phosphorylated by protein kinase A (PKA), inducing proteolysis to Ci-75, a transcriptional repressor. Hh signaling blocks proteolysis and produces an activated Ci-155 transcriptional activator. The relationship between PKA and the Ci/Cos2/Fu complex is unclear. Here we examine Hh target gene expression caused by mutant forms of PKA regulatory (PKAr) and catalytic (PKAc) subunits and by the PKAc inhibitor PKI(1-31). The mutant PKAr*, defective in binding cAMP, is shown to activate Hh target genes solely through its ability to bind and inhibit endogenous PKAc. Surprisingly, PKAcA75, a catalytically impaired mutant, also activates Hh target genes. To account for this observation, we propose that PKAc phosphorylation targeting Ci-155 for proteolysis is regulated within a complex that includes PKAc and Ci-155 and excludes PKI(1-31). This complex may permit processive phosphorylation of Ci-155 molecules, facilitating their processing to Ci-75.

Protein kinase A mediates novel serine-584 phosphorylation of HDAC4

2019 ◽  
Vol 97 (5) ◽  
pp. 526-535 ◽  
Author(s):  
Shanmukha K. Doddi ◽  
Githavani Kummari ◽  
Jagannadham M.V. ◽  
Arunasree M. Kalle
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Cell Line ◽  
Target Genes ◽  
K562 Cells ◽  
Pcr Analysis ◽  
Wild Type ◽  
Kinase A

Given the well-established diversified signaling pathways for histone deacetylase 4 (HDAC4) and the regulation of HDAC4 by several post-translational modifications (PTMs), including phosphorylation, sumoylation, and ubiquitination, an unbiased and detailed analysis of HDAC4 PTMs is needed. In this study, we used matrix-assisted laser desorption/ionization time of flight (MALDI-TOF/TOF) to describe phosphorylation at serine 584 (Ser584) along with already-known dual phosphorylation at serines 265 and 266 (Ser265/266), that together regulate HDAC4 activity. Overexpression of site-specific HDAC4 mutants (S584A, S265/266A) in HEK 293T cells, followed by HDAC activity assays, revealed the mutants to be less active than the wild-type protein. In vitro kinase assays have established that Ser584 and Ser265/266 are phosphorylated by protein kinase A (PKA). Luciferase assays driven by the myocyte enhancer factor 2 (MEF2) promoter and real-time PCR analysis of the MEF2 target genes show that the S584A and S265/266A mutants are less repressive than the wild-type. Furthermore, treatment with PKA activators such as 8-Bromo-cAMP and forskolin, and silencing either by shRNA or its inhibitor H-89 in a mouse myoblast cell line (C2C12) and in a non-muscle human cell line (K562), confirmed in vivo phosphorylation of HDAC4 in C2C12 but not in K562 cells, indicating the specific functional significance of HDAC4 phosphorylation in muscle cells. Thus, we identified PKA-induced Ser584 phosphorylation of HDAC4 as a yet unknown regulatory mechanism of the HDAC4–MEF2 axis.

scholarly journals B56α/Protein Phosphatase 2A Inhibits Adipose Lipolysis in High-Fat Diet-Induced Obese Mice

Endocrinology ◽  
2010 ◽  
Vol 151 (8) ◽  
pp. 3624-3632 ◽  
Author(s):  
Brice P. Kinney ◽  
Liping Qiao ◽  
Justin M. LeVaugh ◽  
Jianhua Shao
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Protein Phosphatase ◽  
High Fat Diet ◽  
Protein Phosphatase 2A ◽  
High Fat ◽  
Protein Levels ◽  
Epididymal Fat ◽  
Phosphatase 2A ◽  
Kinase A

Lipolysis and lipogenesis are two opposite processes that control lipid storage in adipocytes. Impaired adipose lipolysis has been observed in both obese human subjects and animal models. This study investigated the mechanisms underlying impaired adipose lipolysis in a high-fat diet-induced obese (DIO) mouse model. DIO models were created using male C57BL/6 mice. Our results show that β3 adrenergic receptor-specific agonist BRL37344 induced adipose lipolysis was significantly blunted in DIO mice. The levels of Ser660 phosphorylation of hormone-sensitive lipase (HSL) were significantly decreased in the epididymal fat of DIO mice. However, protein levels of HSL, adipose triglyceride lipase and its coactivator comparative gene identification-58 were similar between DIO and control mice. It is known that upon lipolytic hormone stimulation, protein kinase A phosphorylates HSL Ser660 and activates HSL, whereas protein phosphatase 2A (PP2A) dephosphorylates and inactivates HSL. Interestingly, our study shows that high-fat feeding did not alter epididymal fat cAMP and protein kinase A protein levels but significantly increased the expression of the α-isoform of PP2A regulatory subunit B′ (B56α). To study the role of B56α in obesity-associated lipolytic defect, B56α was overexpressed or knocked down by adenovirus-mediated gene transduction in cultured 3T3-L1CARΔ1 adipocytes. Overexpression of B56α significantly decreased HSL Ser660 phosphorylation. In contrast, knocking down B56α increased hormone-stimulated HSL activation and lipolysis in mature 3T3-L1CARΔ1 adipocytes. These results strongly suggest that elevated B56α/PP2A inhibits HSL and lipolysis in white adipose tissue of DIO mice.

scholarly journals Forskolin, 8-Br-3′,5′-Cyclic Adenosine 5′-Monophosphate, and Catalytic Protein Kinase A Expression in the Nucleus Increase Radioiodide Uptake and Sodium/Iodide Symporter Protein Levels in RET/PTC1-Expressing Cells

2004 ◽  
Vol 89 (12) ◽  
pp. 6168-6172 ◽  
Author(s):  
Anjli Venkateswaran ◽  
Derek K. Marsee ◽  
Steven H. Green ◽  
Sissy M. Jhiang
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Sodium Iodide ◽  
Sodium Iodide Symporter ◽  
Thyroid Cells ◽  
Protein Levels ◽  
And Function ◽  
Kinase A

Abstract RET/PTC1, a thyroid-specific oncogene, has been reported to down-regulate sodium/iodide symporter (NIS) expression and function in vitro and in vivo. Recently, RET/PTC1 has been shown to interfere with TSH signaling at multiple levels in thyroid cells. The objective of this study was to investigate whether RET/PTC1-mediated NIS reduction can be rescued by activating cAMP-protein kinase A (PKA) pathways. We showed that both forskolin and 8-Br-cAMP increase radioiodide uptake and NIS protein in RET/PTC1-expressing cells to the same extent as the parental PC Cl 3 cells. We found that RET/PTC1 decreases nuclear localization of catalytic PKA, and forskolin treatment was able to counteract this RET/PTC1 effect. Furthermore, transient expression of catalytic PKA in the nucleus increased radioiodide uptake and NIS protein in RET/PTC1-expressing cells. Taken together, these studies suggest that RET/PTC1 down-regulates NIS expression by interrupting TSH/cAMP signaling, and this RET/PTC1 effect can be reversed by activating cAMP-PKA pathways.

scholarly journals Differential Effects of Protein Kinase A on Ras Effector Pathways

1998 ◽  
Vol 18 (7) ◽  
pp. 3718-3726 ◽  
Author(s):  
Marsha J. Miller ◽  
Lise Rioux ◽  
Gregory V. Prendergast ◽  
Sarah Cannon ◽  
Michael A. White ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Critical Role ◽  
Mitogen Activated Protein Kinase ◽  
Thyroid Cells ◽  
Mapk Phosphorylation ◽  
Differential Effects ◽  
Effector Pathways ◽  
Rat Thyroid ◽  
Kinase A

ABSTRACT Ras mutants with the ability to interact with different effectors have played a critical role in the identification of Ras-dependent signaling pathways. We used two mutants, RasS35 and RasG37, which differ in their ability to bind Raf-1, to examine Ras-dependent signaling in thyroid epithelial cells. Wistar rat thyroid cells are dependent upon thyrotropin (TSH) for growth. Although TSH-stimulated mitogenesis requires Ras, TSH activates protein kinase A (PKA) and downregulates signaling through Raf and the mitogen-activated protein kinase (MAPK) cascade. Cells expressing RasS35, a mutant which binds Raf, or RasG37, a mutant which binds RalGDS, exhibited TSH-independent proliferation. RasS35stimulated morphological transformation and anchorage-independent growth. RasG37 stimulated proliferation but not transformation as measured by these indices. TSH exerted markedly different effects on the Ras mutants and transiently repressed MAPK phosphorylation in RasS35-expressing cells. In contrast, TSH stimulated MAPK phosphorylation and growth in cells expressing RasG37. The Ras mutants, in turn, exerted differential effects on TSH signaling. RasS35 abolished TSH-stimulated changes in cell morphology and thyroglobulin expression, while RasG37 had no effect on these activities. Together, the data indicate that cross talk between Ras and PKA discriminates between distinct Ras effector pathways.

scholarly journals Progesterone activates the cyclic AMP-protein kinase A signalling pathway by upregulating ABHD2 in fertile men

2021 ◽  
Vol 49 (3) ◽  
pp. 030006052199952
Author(s):  
Feng Jiang ◽  
Yong Zhu ◽  
Ying Chen ◽  
Xiaofeng Tang ◽  
Liu Liu ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Western Blot ◽  
Protein Kinase A ◽  
Gene Sequencing ◽  
Signalling Pathway ◽  
Protein Levels ◽  
Infertile Men ◽  
A Prospective Study ◽  
Kinase A

Objective This was a prospective study to investigate whether progesterone affects sperm activity by regulating the cyclic AMP-protein kinase A (cAMP-PKA) signalling pathway via α/β hydrolase domain-containing protein 2 (ABHD2). Methods Spermatozoa were collected from healthy and infertile men (with oligoasthenospermia or abnormal acrosome; n = 30/group). The expression of and mutations in ABHD2 were detected by quantitative PCR, western blot, and gene sequencing. The expression of ABHD2 in the presence of progesterone was detected in all groups, and cAMP and PKA levels were detected by ELISA in fertile men after treatment with ABHD2 antibody and PKA inhibitor H-89, respectively. Results Expression of ABHD2 mRNA and protein were reduced in spermatozoa from infertile compared with fertile men. Four gene mutation sites were detected in spermatozoa from the infertile groups. Progesterone increased mRNA and protein levels of ABHD2 in healthy spermatozoa but not in spermatozoa from infertile men. The levels of cAMP and PKA were increased by progesterone in healthy spermatozoa, and the progesterone-increased cAMP and PKA were decreased by ABHD2 antibody and H-89, respectively. Conclusion Progesterone regulates the ABHD2-mediated cAMP-PKA signalling pathway in healthy spermatozoa, which provides a new target for clinical diagnosis and treatment of infertility.

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