Consequences of Lysine 72 Mutation on the Phosphorylation and Activation State of cAMP-dependent Kinase

Iyer, Ganesh H.; Moore, Michael J.; Taylor, Susan S.

doi:10.1074/jbc.m407586200

Cited by 70 publications

(74 citation statements)

References 44 publications

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“…These results are consistent with Ser 338 phosphorylation occurring cotranslationally by a cis mechanism as a one-off event, meaning that it happens one time during translation. The order of Ser 338 and Thr 197 phosphorylation that we observe differs from the order described in a previous report (21), but in that report, H-89, an inhibitor of C-subunit, was used to generate unphosphorylated C-subunit (21); additionally, the work by Iyer et al (21) monitored the kinetics of phosphorylation at Thr 197 by PDK1 and Ser 338 by autophosphorylation, which contrasts with our approach that involved assessment of the kinetics of phosphorylation in parallel with translation of the C-subunit. Because Ser 338 phosphorylation is unique to PKA in the AGC subfamily (Fig.…”

Section: Ser 338 Phosphorylation Precedes Thr 197 Phosphorylation and Iscontrasting

confidence: 56%

“…Similarly, the C R336A mutant cannot be autophosphorylated at Ser 338 . Mutation of the catalytic lysine, C K72H , generates a protein that is not phosphorylated at either Thr 197 or Ser 338 (21). We expressed all three mutants in E. coli, purified them to homogeneity along with WT C-subunit, and then assessed their phosphorylation state and enzymatic activity (Fig.…”

Section: Resultsmentioning

confidence: 99%

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Cotranslational cis -phosphorylation of the COOH-terminal tail is a key priming step in the maturation of cAMP-dependent protein kinase

Keshwani¹,

Klammt

Daake

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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cAMP-dependent protein kinase A (PKA), ubiquitously expressed in mammalian cells, regulates a plethora of cellular processes through its ability to phosphorylate many protein substrates, including transcription factors, ion channels, apoptotic proteins, transporters, and metabolic enzymes. The PKA catalytic subunit has two phosphorylation sites, a well-studied site in the activation loop (Thr 197 ) and another site in the C-terminal tail (Ser 338 ) for which the role of phosphorylation is unknown. We show here, using in vitro studies and experiments with S49 lymphoma cells, that cis-autophosphorylation of Ser 338 occurs cotranslationally, when PKA is associated with ribosomes and precedes posttranslational phosphorylation of the activation loop Thr 197 . Ser 338 phoshorylation is not required for PKA activity or formation of the holoenzyme complex; however, it is critical for processing and maturation of PKA, and it is a prerequisite for phosphorylation of Thr 197 . After Thr 197 and Ser 338 are phosphorylated, both sites are remarkably resistant to phosphatases. Phosphatase resistance of the activation loop, a unique feature of both PKA and PKG, reflects the distinct way that signal transduction dynamics are controlled by cyclic nucleotide-dependent PKs.

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Section: Ser 338 Phosphorylation Precedes Thr 197 Phosphorylation and Iscontrasting

confidence: 56%

Section: Resultsmentioning

confidence: 99%

Cotranslational cis -phosphorylation of the COOH-terminal tail is a key priming step in the maturation of cAMP-dependent protein kinase

Keshwani¹,

Klammt

Daake

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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“…It certainly disrupts the initial autophosphorylation event on Thr 197 that leads to secondary phosphorylations in the C-subunit when the enzyme is expressed in E. coli. Phosphorylation of Ser 338 is thought to be intramolecular (20).…”

Section: Discussionmentioning

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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“…The activation loop is a flexible loop spatially close to the catalytic loop. Activation loop phosphorylation can counteract the positive charge of the arginine in the catalytic loop by the HRD motif (Iyer et al 2005, Steichen et al 2010, which in the case of RET this is commonly seen by a salt bridge network between αC R770 and activation loop R897 and K907, Fig. 2 and (Knowles et al 2006, Plaza-Menacho et al 2014a.…”

Section: Conserved Structural and Functional Features Of Ret Catalytimentioning

confidence: 99%

Structure and function of RET in multiple endocrine neoplasia type 2

Plaza-Menacho¹

2018

Endocrine-Related Cancer

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Correspondence should be addressed to I Plaza-Menacho: iplaza@cnio.esThis paper is part of a thematic review section on 25 Years of RET and MEN2. The guest editors for this section were Lois Mulligan and Frank Weber. AbstractIt has been twenty-five years since the discovery of oncogenic germline RET mutations as the cause of multiple endocrine neoplasia type 2 (MEN2). Intensive work over the last two and a half decades on RET genetics, signaling and cell biology has provided the current bases for the genotype-phenotype and functional correlations within this cancer syndrome. On the contrary, the structural and molecular basis for RET tyrosine kinase domain activation and oncogenic deregulation has remained largely elusive. Recent studies with a strong crystallographic and biochemical focus have started to elucidate key insights into such molecular and atomic details revealing unexpected and private mechanisms of actions and molecular determinants not previously envisioned. This review focuses on the structure and function of the RET receptor, and in particular, on what a more detailed view of the protein itself and what the current structural and molecular information tell us about the genotype and phenotype relationships in the cancer syndrome MEN2.

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Consequences of Lysine 72 Mutation on the Phosphorylation and Activation State of cAMP-dependent Kinase

Abstract: General strategies to obtain inactive kinases have utilized mutation of key conserved residues in the kinase core, and the equivalent Lys 72 in cAMP-dependent kinase has often been used to generate a "dead" kinase.

Cited by 70 publications

References 44 publications

Cotranslational cis -phosphorylation of the COOH-terminal tail is a key priming step in the maturation of cAMP-dependent protein kinase

Cotranslational cis -phosphorylation of the COOH-terminal tail is a key priming step in the maturation of cAMP-dependent protein kinase

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

Structure and function of RET in multiple endocrine neoplasia type 2

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