Phosphorylation of the Catalytic Subunit of Protein Kinase A

Moore, Michael J.; Kanter, Joan R.; Jones, Kristian; Taylor, Susan S.

doi:10.1074/jbc.m204970200

Cited by 113 publications

(54 citation statements)

References 37 publications

(43 reference statements)

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“…The blots were probed with an anti-phosphothreonine 197 antibody (kindly provided by A. Newton, UCSD) and anti-phosphoserine 338 antibody (SynPep Corp.), a custom antibody described previously (12). The secondary antibody was an anti-rabbit horseradish peroxidase (HRP) conjugate (Amersham Biosciences).…”

Section: Methodsmentioning

confidence: 99%

Identification of ChChd3 as a Novel Substrate of the cAMP-dependent Protein Kinase (PKA) Using an Analog-sensitive Catalytic Subunit

Schauble

King

Darshi

et al. 2007

Journal of Biological Chemistry

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Due to the numerous kinases in the cell, many with overlapping substrates, it is difficult to find novel substrates for a specific kinase. To identify novel substrates of cAMP-dependent protein kinase (PKA), the PKA catalytic subunit was engineered to accept bulky N 6 -substituted ATP analogs, using a chemical genetics approach initially pioneered with v-Src (1). Methionine 120 was mutated to glycine in the ATP-binding pocket of the catalytic subunit. To express the stable mutant C-subunit in Escherichia coli required co-expression with PDK1. This mutant protein was active and fully phosphorylated on Thr 197 and Ser 338 . Based on its kinetic properties, the engineered C-subunit preferred N 6 (benzyl)-ATP and N 6 (phenethyl)-ATP over other ATP analogs, but still retained a 30 M K m for ATP. This mutant recombinant C-subunit was used to identify three novel PKA substrates. One protein, a novel mitochondrial ChChd protein, ChChd3, was identified, suggesting that PKA may regulate mitochondria proteins.The cAMP-dependent protein kinase (PKA) 3 is expressed in all mammalian cells and plays a major role in processes such as metabolic control, memory, growth, development, and apoptosis. Thus, it has many substrates, which usually are highly tissue specific. One such example is the bifunctional enzyme phosphofructokinase-2 (PFK2), which upon phosphorylation by PKA is converted to its phosphatase active mode in liver, but is stabilized in its kinase mode in heart, and is not a PKA substrate in skeletal muscle (1). Although PKA has been widely studied in most cells, the direct substrates of PKA are difficult to distinguish from downstream substrates that are phosphorylated as a result of the PKA activation. A possible way to determine whether identified substrates are modified by the kinase directly or indirectly via an intermediary kinase is to engineer the ATP-binding pocket of the kinase in question to accept a bulky ATP analog that cannot be used by the wild type kinase. This strategy was developed initially for a tyrosine kinase, v-Src, (2) and has subsequently been used for other protein kinases (3). Shah et al. (4) found that a single mutation conferred specificity for ATP analogs that have a large substituent on the nitrogen at position 6 (N 6 ) on the purine ring of ATP. Two residues that were found within 5 Å of the N 6 position of ATP were based initially on the structures of PKA and CDK2 (cyclindependent kinase 2) since the crystal structure of v-Src was not available at the time (2). In v-Src, these residues are Val 323 and Ile 338 , while the corresponding residues in PKA are Val 104 and Met 120 . Although the Val 323 mutation was found to be not important for conferring specificity in v-Src, mutation of Ile 338 to Ala did alter the specificity of v-Src and allowed the enzyme to use an orthogonal bulky ATP analog. Once the crystal structure of hematopoietic cell kinase (Hck), a Src family tyrosine kinase was available, molecular modeling was employed to explore more fully the binding pocket of Hck to find t...

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Section: Methodsmentioning

confidence: 99%

Identification of ChChd3 as a Novel Substrate of the cAMP-dependent Protein Kinase (PKA) Using an Analog-sensitive Catalytic Subunit

Schauble

King

Darshi

et al. 2007

Journal of Biological Chemistry

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“…However, the kinase responsible for this phosphorylation has never been identified. The corresponding site in mammalian PKA is also phosphorylated, and for many years, the upstream kinase was not known with certainty (30,32). PKA autophosphorylation and PDK1-dependent phosphorylation are the two main candidate mechanisms.…”

Section: Plasmid (Prs316)mentioning

confidence: 99%

Yeast 3-Phosphoinositide-dependent Protein Kinase-1 (PDK1) Orthologs Pkh1–3 Differentially Regulate Phosphorylation of Protein Kinase A (PKA) and the Protein Kinase B (PKB)/S6K Ortholog Sch9

Voordeckers¹,

Kimpe

Haesendonckx

et al. 2011

Journal of Biological Chemistry

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Pkh1, -2, and -3 are the yeast orthologs of mammalian 3-phosphoinositide-dependent protein kinase-1 (PDK1). Although essential for viability, their functioning remains poorly understood. Sch9, the yeast protein kinase B and/or S6K ortholog, has been identified as one of their targets. We now have shown that in vitro interaction of Pkh1 and Sch9 depends on the hydrophobic PDK1-interacting fragment pocket in Pkh1 and requires the complementary hydrophobic motif in Sch9. We demonstrated that Pkh1 phosphorylates Sch9 both in vitro and in vivo on its PDK1 site and that this phosphorylation is essential for a wild type cell size. In vivo phosphorylation on this site disappeared during nitrogen deprivation and rapidly increased again upon nitrogen resupplementation. In addition, we have shown here for the first time that the PDK1 site in protein kinase A is phosphorylated by Pkh1 in vitro, that this phosphorylation is Pkhdependent in vivo and occurs during or shortly after synthesis of the protein kinase A catalytic subunits. Mutagenesis of the PDK1 site in Tpk1 abolished binding of the regulatory subunit and cAMP dependence. As opposed to PDK1 site phosphorylation of Sch9, phosphorylation of the PDK1 site in Tpk1 was not regulated by nitrogen availability. These results bring new insight into the control and prevalence of PDK1 site phosphorylation in yeast by Pkh protein kinases.

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“…The activity of PKA catalytic subunits (PKAcs) is regulated not only by the binding of cAMP onto regulatory subunit, but also by their phosphorylation at Thr197, which is autophosphorylated or phosphorylated by phosphoinositide‐dependent kinase‐1 (Moore et al ., 2002). For maximal activity, each catalytic subunit must also be either autophosphorylated or phosphorylated at Thr197, which helps orient catalytic residues in the active site (Steichen et al ., 2010, 2012).…”

Section: Introductionmentioning

confidence: 99%

O‐GlcNAcylation of protein kinase A catalytic subunits enhances its activity: a mechanism linked to learning and memory deficits in Alzheimer's disease

Xie

Jin

et al. 2016

Aging Cell

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SummaryAlzheimer's disease (AD) is characterized clinically by memory loss and cognitive decline. Protein kinase A (PKA)‐CREB signaling plays a critical role in learning and memory. It is known that glucose uptake and O‐GlcNAcylation are reduced in AD brain. In this study, we found that PKA catalytic subunits (PKAcs) were posttranslationally modified by O‐linked N‐acetylglucosamine (O‐GlcNAc). O‐GlcNAcylation regulated the subcellular location of PKAcα and PKAcβ and enhanced their kinase activity. Upregulation of O‐GlcNAcylation in metabolically active rat brain slices by O‐(2‐acetamido‐2‐deoxy‐d‐glucopyranosylidenamino) N‐phenylcarbamate (PUGNAc), an inhibitor of N‐acetylglucosaminidase, increased the phosphorylation of tau at the PKA site, Ser214, but not at the non‐PKA site, Thr205. In contrast, in rat and mouse brains, downregulation of O‐GlcNAcylation caused decreases in the phosphorylation of CREB at Ser133 and of tau at Ser214, but not at Thr205. Reduction in O‐GlcNAcylation through intracerebroventricular injection of 6‐diazo‐5‐oxo‐l‐norleucine (DON), the inhibitor of glutamine fructose‐6‐phosphate amidotransferase, suppressed PKA‐CREB signaling and impaired learning and memory in mice. These results indicate that in addition to cAMP and phosphorylation, O‐GlcNAcylation is a novel mechanism that regulates PKA‐CREB signaling. Downregulation of O‐GlcNAcylation suppresses PKA‐CREB signaling and consequently causes learning and memory deficits in AD.

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Phosphorylation of the Catalytic Subunit of Protein Kinase A

Cited by 113 publications

References 37 publications

Identification of ChChd3 as a Novel Substrate of the cAMP-dependent Protein Kinase (PKA) Using an Analog-sensitive Catalytic Subunit

Identification of ChChd3 as a Novel Substrate of the cAMP-dependent Protein Kinase (PKA) Using an Analog-sensitive Catalytic Subunit

Yeast 3-Phosphoinositide-dependent Protein Kinase-1 (PDK1) Orthologs Pkh1–3 Differentially Regulate Phosphorylation of Protein Kinase A (PKA) and the Protein Kinase B (PKB)/S6K Ortholog Sch9

O‐GlcNAcylation of protein kinase A catalytic subunits enhances its activity: a mechanism linked to learning and memory deficits in Alzheimer's disease

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