Molecular basis for isoform-specific autoregulation of protein kinase A

Diskar, Mandy; Zenn, Hans-Michael; Kaupisch, Alexandra; Prinz, Anke; Herberg, Friedrich W.

doi:10.1016/j.cellsig.2007.05.012

Cited by 34 publications

(51 citation statements)

References 36 publications

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“…Cloning of human PRKAR1A, PRKAR2A, PRKACA, and PRKX genes into the BRET vectors was published previously (15,16). Mutagenesis of PrKX was performed using plasmid pFastBac HTb-PRKX (15) as the template.…”

Section: Methodsmentioning

confidence: 99%

“…Point mutations of C␣, PrKX, F47F2.1b, PKAC3, RI␤, and RII␤ were introduced using the oligonucleotides listed under supplemental Table S2. Mutagenesis of RI␣ and RII␣ was published previously (15). All plasmids used in this study were sequence verified.…”

Section: Methodsmentioning

confidence: 99%

“…His 6 -tagged human C␣ (His 6 -C␣) and His 6 -C␣ L277R were expressed overnight at room temperature in Escherichia coli BL21(DE3) (Novagen) and purified using a Talon affinity resin and standard conditions (Clontech). Expression and purification of His 6 -PrKX and His 6 -PrKX R283L was performed as described (15).…”

Section: Methodsmentioning

confidence: 99%

“…Previously, we identified the R subunit autoinhibitory site as a main determinant for isoform-specific regulation of PKA (15). We were able to gain significant binding of PrKX with RII␣ solely by mutation of Ser-99 to Ala in the RII␣ autoinhibitory domain (P 0 -site).…”

mentioning

confidence: 98%

“…It was found to be crucial for macrophage and granulocyte maturation (9, 10). PrKX was shown to be involved in renal development, regulating epithelial cell migration, ureteric bud branching, and induction of glomeruli formation (8,(11)(12)(13).C␣ and human PrKX differ by their selective holoenzyme formation in living cells, as PrKX is inhibited only by RI␣, but not by RII␣ (14,15). Here, we have tested all four human R subunits for the first time side by side and show that this so far unique property of RI over RII preference with respect to autoinhibition appears to be an evolutionarily conserved feature of PrKX and at least four of its orthologs (Mus musculus Pkare, Drosophila melanogaster DC2, Trypanosoma brucei PKAC3, human PrKY), and possibly also Caenorhabditis elegans F47F2.1b.…”

mentioning

confidence: 99%

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Regulation of cAMP-dependent Protein Kinases

Diskar

Zenn

Kaupisch

et al. 2010

Journal of Biological Chemistry

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cAMP-dependent protein kinases are reversibly complexed with any of the four isoforms of regulatory (R) subunits, which contain either a substrate or a pseudosubstrate autoinhibitory domain. The human protein kinase X (PrKX) is an exemption as it is inhibited only by pseudosubstrate inhibitors, i.e. RI␣ or RI␤ but not by substrate inhibitors RII␣ or RII␤. Detailed examination of the capacity of five PrKX-like kinases ranging from human to protozoa (Trypanosoma brucei) to form holoenzymes with human R subunits in living cells shows that this preference for pseudosubstrate inhibitors is evolutionarily conserved. To elucidate the molecular basis of this inhibitory pattern, we applied bioluminescence resonance energy transfer and surface plasmon resonance in combination with site-directed mutagenesis. We observed that the conserved ␣H-␣I loop residue Arg-283 in PrKX is crucial for its RI over RII preference, as a R283L mutant was able to form a holoenzyme complex with wild type RII subunits. Changing the corresponding ␣H-␣I loop residue in PKA C␣ (L277R), significantly destabilized holoenzyme complexes in vitro, as cAMP-mediated holoenzyme activation was facilitated by a factor of 2-4, and lead to a decreased affinity of the mutant C subunit for R subunits, significantly affecting RII containing holoenzymes.The protein kinase A (PKA) holoenzyme is a heterotetramer composed of two catalytic (C) 5 subunits kept inactive by a dimer of R subunits. Each R subunit monomer contains two tandem cAMP-binding domains, in which the sequential binding of two cAMP molecules releases an active C subunit (1). Two main classes of PKA isozymes, type I and type II, distinguishable by their R subunits, have been described. Crystal structures and solution scattering data provide evidence of a complex interaction network between C and R subunits as well as differences in global structure of PKA type I and type II holoenzymes (2-5).Homo sapiens express the PKA R subunit isoforms RI␣, RI␤, RII␣, and RII␤ and C subunits C␣, C␤, and C␥. Another cAMPdependent protein kinase is PrKX and possibly protein kinase Y (PrKY). PrKY is 94% homologous to PrKX, but shortened by 81 amino acids at the C terminus. On the genome level, PRKX and PRKY are implicated in sex-reversal disorders (6). PrKX is being discussed as a phylogenetically and functionally separate enzyme (7,8). In contrast to the ubiquitously expressed C␣ subunit, PrKX is mainly active during embryonic organ development and cellular differentiation in hematopoietic lineages. It was found to be crucial for macrophage and granulocyte maturation (9, 10). PrKX was shown to be involved in renal development, regulating epithelial cell migration, ureteric bud branching, and induction of glomeruli formation (8,(11)(12)(13).C␣ and human PrKX differ by their selective holoenzyme formation in living cells, as PrKX is inhibited only by RI␣, but not by RII␣ (14,15). Here, we have tested all four human R subunits for the first time side by side and show that this so far unique property of RI over RII pr...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 98%

mentioning

confidence: 99%

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Regulation of cAMP-dependent Protein Kinases

Diskar

Zenn

Kaupisch

et al. 2010

Journal of Biological Chemistry

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PRKX, a Novel cAMP-Dependent Protein Kinase Member, Plays an Important Role in Development

Huang

Alberts

et al. 2016

J. Cell. Biochem.

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The human protein kinase X gene (PRKX) and cAMP-dependent protein kinase (PKA) are both c-AMP-dependent serine/threonine protein kinases within the protein kinase AGC subgroup. Of all the protein kinases in this group, PRKX is the least studied. PRKX has been isolated from patients with chondrodysplasia punctate and is involved in numerous processes, including sexual differentiation and fertilization, normal kidney development and autosomal dominant polycystic kidney disease (ADPKD), blood maturation, neural development, and angiogenesis in vitro. Although the role of PRKX in development and disease has been reported recently, the underlying mechanism of PRKX activity is largely unknown. In addition, based on the expression pattern of PRKX and the extensive role of PKA in disease and development, PRKX might have additional crucial functions that have not been addressed in the literature. In this review, we summarize the characteristics and developmental functions of PRKX that have been reported by recent studies. In particular, we elucidate the structural and functional differences between PRKX and PKA, as well as the possible roles of PRKX in development and related diseases. Finally, we propose future studies that could lead to important discoveries of more PRKX functions and the underlying mechanisms involved.

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Survey of the year 2007 commercial optical biosensor literature

Rich

Myszka

2008

J of Molecular Recognition

163

121

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In 2007, 1179 papers were published that involved the application of optical biosensors. Reported developments in instrument hardware, assay design, and immobilization chemistry continue to improve the technology's throughput, sensitivity, and utility. Compared to recent years, the widest range of platforms, both traditional format and array-based, were used. However, as in the past, we found a disappointingly low percentage of well-executed experiments and thoughtful data interpretation. We are alarmed by the high frequency of suboptimal data and over-interpreted results in the literature. Fortunately, learning to visually recognize good--and more importantly, bad--data is easy. Using examples from the literature, we outline several features of biosensor responses that indicate experimental artifacts versus actual binding events. Our goal is to have everyone, from benchtop scientists to project managers and manuscript reviewers, become astute judges of biosensor results using nothing more than their eyes.

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Molecular basis for isoform-specific autoregulation of protein kinase A

Cited by 34 publications

References 36 publications

Regulation of cAMP-dependent Protein Kinases

Regulation of cAMP-dependent Protein Kinases

PRKX, a Novel cAMP-Dependent Protein Kinase Member, Plays an Important Role in Development

Survey of the year 2007 commercial optical biosensor literature

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