PDK1 acquires PDK2 activity in the presence of a synthetic peptide derived from the carboxyl terminus of PRK2

Balendran, Anudharan; Casamayor, Antonio; Deák, Mária; Paterson, Andrew J.; Gaffney, Piers R. J.; Currie, Richard A.; Downes, C P; Alessi, Dario R.

doi:10.1016/s0960-9822(99)80186-9

Cited by 413 publications

(355 citation statements)

References 46 publications

Supporting

Mentioning

331

Contrasting

Unclassified

Order By: Relevance

“…PDK1 is also able to phosphorylate PKBα at Ser-473 in a PtdIns(3,4,5)P $ -dependent manner when it is complexed to a peptide comprising the 24 C-terminal amino acids of PRK2 [9]. The GST-S25A-PDK1, GST-S393A\396A-PDK1 and GST-S410A-PDK1 mutants were also capable of phosphorylating Ser-473 of PKBα in the presence the PRK2 C-terminal peptide,…”

Section: Role Of the Pdk1 Phosphorylation Sites In Regulating Pdk1 Acmentioning

confidence: 98%

“…The indicated GST-PDK1 proteins (20 ng) were incubated in the presence (j) or absence (k) of 100 µM of the PRK2 C-terminal peptide (corresponding to residues 969-984 of human PRK2 [9]). After standing for 1 h at 4 mC, GST-PKBα, MgATP and phospholipid vesicles containing 100 µM phosphatidylcholine, 100 µM phosphatidylserine and 10 µM sn-1-stearoyl-2-arachidonyl-D-PtdIns(3,4,5)P 3 were added.…”

Section: Figure 5 Effect Of Phosphorylation-site Mutants On the Rate mentioning

confidence: 99%

“…Phosphorylation of both the PDK1 and PDK2 sites is required for maximal activation of PKB or p70 S6 kinase and\or stability for PKC isoforms. Recent observations have demonstrated that PDK1 can acquire the ability to phosphorylate the PDK2 site on PKB as well as Thr-308 in the presence of a synthetic peptide derived from the C-terminus of PKC-related kinase-2 (PRK2) [9]. These observations suggest that PDK1 and PDK2 may be the same enzyme.…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Phosphorylation of Ser-241 is essential for the activity of 3-phosphoinositide-dependent protein kinase-1: identification of five sites of phosphorylation in vivo

Casamayor

Morrice

Alessi

1999

Biochemical Journal

Self Cite

281

127

View full text Add to dashboard Cite

3-Phosphoinositide-dependent protein kinase-1 (PDK1) expressed in unstimulated 293 cells was phosphorylated at Ser-25, Ser-241, Ser-393, Ser-396 and Ser-410 and the level of phosphorylation of each site was unaffected by stimulation with insulin-like growth factor-1. Mutation of Ser-241 to Ala abolished PDK1 activity, whereas mutation of the other phosphorylation sites individually to Ala did not affect PDK1 activity. Ser-241, unlike the other phosphorylation sites on PDK1, was resistant to dephosphorylation by protein phosphatase 2A1. Ser-241 lies in the activation loop of the PDK1 kinase domain between subdomains VII and VIII in the equivalent position to the site that PDK1 phosphorylates on its protein kinase substrates. PDK1 expressed in bacteria was active and phosphorylated at Ser-241, suggesting that PDK1 can phosphorylate itself at this site, leading to its own activation.

show abstract

Section: Role Of the Pdk1 Phosphorylation Sites In Regulating Pdk1 Acmentioning

confidence: 98%

Section: Figure 5 Effect Of Phosphorylation-site Mutants On the Rate mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Phosphorylation of Ser-241 is essential for the activity of 3-phosphoinositide-dependent protein kinase-1: identification of five sites of phosphorylation in vivo

Casamayor

Morrice

Alessi

1999

Biochemical Journal

Self Cite

281

127

View full text Add to dashboard Cite

show abstract

“…The identity of the kinase(s) responsible for phosphorylating Ser-473 (provisionally referred to as 'PDK2') has remained enigmatic, but recent work by Sarbassov et al (2005) has provided compelling genetic and biochemical evidence indicating that the rapamycininsensitive mammalian target of rapamycin complex (mTORC2) mediates this event. Other kinases have been implicated as 'PDK2', including PDK1 (Balendran et al, 1999), DNA-PK (Feng et al, 2004), ILK (Persad et al, 2001) and Akt itself (Toker and Newton, 2000).…”

Section: Introductionmentioning

confidence: 99%

Akt inhibitor A-443654 induces rapid Akt Ser-473 phosphorylation independent of mTORC1 inhibition

et al. 2007

View full text Add to dashboard Cite

Rapamycin, a natural product inhibitor of the Raptormammalian target of rapamycin complex (mTORC1), is known to induce Protein kinase B (Akt/PKB) Ser-473 phosphorylation in a subset of human cancer cell lines through inactivation of S6K1, stabilization of insulin receptor substrate (IRS)-1, and increased signaling through the insulin/insulin-like growth factor-I/phosphatidylinositol 3-kinase (PI3K) axis. We report that A-443654, a potent small-molecule inhibitor of Akt serine/threonine kinases, induces Akt Ser-473 phosphorylation in all human cancer cell lines tested, including PTEN-and TSC2-deficient lines. This phenomenon is dose-dependent, manifests coincident with Akt inhibition and likely represents an alternative, rapid-feedback pathway that can be functionally dissociated from mTORC1 inhibition. Experiments performed in TSC2 À/À cells indicate that TSC2 and IRS-1 cooperate with, but are dispensable for, A-443654-mediated Akt phosphorylation. This feedback event does require PI3K activity, however, as it can be inhibited by LY294002 or wortmannin. Small interfering RNA-mediated knockdown of mTOR or Rictor, components of the rapamycin-insensitive mTORC2 complex, but not the mTORC1 component Raptor, also inhibited Akt Ser-473 phosphorylation induced by A-443654. Our data thus indicate that Akt phosphorylation and activity are coupled in a manner not previously appreciated and provide a novel mode of Akt regulation that is distinct from the previously described rapamycin-induced IRS-1 stabilization mechanism.

show abstract

“…However, Src, Fyn, and Abl tyrosine kinases have been shown to phosphorylate PDK1 in vitro and overexpression of v-Src in mammalian cells results in tyrosine phosphorylation and activation of PDK1 [15][16][17]. In addition, mammalian PDK1 is affected by interactions with PDK1-associating proteins, including PKC-related kinase 1 (PRK1), and Hsp90 [18][19][20][21], suggesting that its activity is controlled by interactions with other proteins as well. Furthermore, human PDK1 has been shown to interact with 14-3-3 h and g and a 14-3-3 recognition site has been identified at Ser-241 [22].…”

Section: Introductionmentioning

confidence: 99%

Arabidopsis PDK1: identification of sites important for activity and downstream phosphorylation of S6 kinase

et al. 2006

View full text Add to dashboard Cite

The Arabidopsis thaliana protein kinase AtPDK1 was identified as a homologue of the mammalian 3-phosphoinositide-dependent protein kinase-1 (PDK1), which is involved in a number of physiological processes including cell growth and proliferation. We now show that AtPDK1, expressed in E. coli as a recombinant protein, undergoes autophosphorylation at several sites. Using mass spectrometry, three phosphorylated amino acid residues, Ser-177, Ser-276 and Ser-382, were identified, followed by mutational analyses to reveal their roles. These residues are not conserved in mammalian PDK1s. Mutation of Ser-276 in AtPDK1 to alanine resulted in an enzyme with no detectable autophosphorylation. Autophosphorylation was significantly reduced in the Ser177Ala mutant but was only slightly reduced in the Ser382Ala mutant. Other identified sites of importance for autophosphorylation and/or activity of AtPDK1 were Asp-167, Thr-176, and Thr-211. Sites in the mammalian PDK1 corresponding to Asp-167 and Thr-211 are essential for PDK1 autophosphorylation and activity. Autophosphorylation was absent in the Asp167Ala mutant while the Thr176Ala and The211Ala mutants exhibited very low but detectable autophosphorylation, pointing to both similarity and difference between mammalian and plant enzymes. We also demonstrate that AtS6k2, an A. thaliana homologue to the mammalian S6 kinases, is an in vitro target of AtPDK1. Our data clearly show that Asp-167, Thr-176, Ser-177, Thr-211, and Ser-276 in AtPDK1 are important for the downstream phosphorylation of AtS6k2. The results confirm that AtPDK1, like mammalian PDK1, needs phosphorylation at several sites for full downstream phosphorylation activity. Finally, we investigated A. thaliana 14-3-3 proteins as potential AtPDK1 regulatory proteins and the effect of phospholipids on the AtPDK1 activity. Nine of the 12 14-3-3 isoforms tested enhanced AtPDK1 activity whereas one isoform suppressed the activity. No significant effects on AtPDK1 activity by the various phospholipids (including phosphoinositides) were evident.

show abstract

PDK1 acquires PDK2 activity in the presence of a synthetic peptide derived from the carboxyl terminus of PRK2

Abstract: PDK1 and PDK2 might be the same enzyme, the substrate specificity and activity of PDK1 being regulated through its interaction with another protein(s). PRK2 is a probable substrate for PDK1.

Cited by 413 publications

References 46 publications

Phosphorylation of Ser-241 is essential for the activity of 3-phosphoinositide-dependent protein kinase-1: identification of five sites of phosphorylation in vivo

Phosphorylation of Ser-241 is essential for the activity of 3-phosphoinositide-dependent protein kinase-1: identification of five sites of phosphorylation in vivo

Akt inhibitor A-443654 induces rapid Akt Ser-473 phosphorylation independent of mTORC1 inhibition

Arabidopsis PDK1: identification of sites important for activity and downstream phosphorylation of S6 kinase

Contact Info

Product

Resources

About