Identification of a pocket in the PDK1 kinase domain that interacts with PIF and the C-terminal residues of PKA

Biondi, Ricardo M.; Cheung, Pierina; Casamayor, Antonio; Deák, Mária; Currie, Richard A.; Alessi, Dario R.

doi:10.1093/emboj/19.5.979

Cited by 290 publications

(384 citation statements)

References 51 publications

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“…This PH domain binds to PI(3,4,5)P3. PDK1 binds to an FXXF motif on the C-terminal tail of PKA (23) and its other substrates. PDK1 then phosphorylates PKA at Thr 197 .…”

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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“…This PH domain binds to PI(3,4,5)P3. PDK1 binds to an FXXF motif on the C-terminal tail of PKA (23) and its other substrates. PDK1 then phosphorylates PKA at Thr 197 .…”

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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“…PKAc has been shown to interact directly with several proteins including the NF-B inhibitory proteins I B-␣ and I B-␤ (37), the transcription factor serum amyloid A-activating factor-1 (38), and 3-phosphoinositide-dependent kinase-1 (PDK1) (39). The interaction with PDK1 is intriguing because it is mediated by a hydrophobic motif at the C terminus of PKAc, which is homologous to the PDK1-interacting motif of several PDK1 substrates (39).…”

Section: Discussionmentioning

confidence: 99%

“…The interaction with PDK1 is intriguing because it is mediated by a hydrophobic motif at the C terminus of PKAc, which is homologous to the PDK1-interacting motif of several PDK1 substrates (39). PDK1 can also activate PKA by phosphorylation of Thr-197 in the activation loop (40), but stable association between PDK1 and full-length PKAc has not been demonstrated (only the C-terminal 223 aa of PKA were used in ref.…”

Section: Discussionmentioning

confidence: 99%

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Inhibition of Xenopus oocyte meiotic maturation by catalytically inactive protein kinase A

Schmitt

Nebreda

2002

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

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Progesterone induces G2-arrested Xenopus oocytes to develop into fertilizable eggs in a process called meiotic maturation. Protein kinase A (PKA), the cAMP-dependent protein kinase, has long been known to be a potent inhibitor of meiotic maturation, but little information is available on how PKA functions. We have cloned two Xenopus PKA catalytic subunit isoforms, XPKA␣ and XPKA␤. These proteins are 89% identical and both inhibit progesteroneinduced meiotic maturation when overexpressed at low levels, suggesting that PKA activity is tightly regulated in the oocyte. Unexpectedly, catalytically inactive XPKA mutants are able to block progesterone-induced maturation as efficiently as the wild-type active XPKA. These mutants also block meiotic maturation induced by Mos, but are less efficient at inhibiting Cdc25C-induced maturation. Our results indicate that PKA can inhibit meiotic maturation by a novel mechanism, which does not require its kinase activity and is also independent of binding to the PKA regulatory subunits.

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“…The elucidation of the three-dimensional cocrystal structures of PKA, Akt and PDK1 bound to ATP, ATP analogs or kinase inhibitors (Biondi et al, 2000;Yang et al, 2002a;Komander et al, 2004) may provide initial insights into how PDK1 kinase selectivity might be achieved with ATP-competitive inhibitors. We can expect that these structure-based design efforts combined with new screening methods (for example, inverse in silico screening; Zahler et al, 2007) may provide the basis for the identification and development of a new generation of PDK1 modulators with the desirable activity/selectivity profile and pharmacological properties.…”

Section: Inhibitors Of the Ser/thr Kinase Activity Of Pdk1mentioning

confidence: 99%

Drug discovery approaches targeting the PI3K/Akt pathway in cancer

2008

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Identification of a pocket in the PDK1 kinase domain that interacts with PIF and the C-terminal residues of PKA

Cited by 290 publications

References 51 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

Inhibition of Xenopus oocyte meiotic maturation by catalytically inactive protein kinase A

Drug discovery approaches targeting the PI3K/Akt pathway in cancer

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