Further evidence that 3-phosphoinositide-dependent protein kinase-1 (PDK1) is required for the stability and phosphorylation of protein kinase C (PKC) isoforms.
The multi-site phosphorylation of the protein kinase C (PKC) superfamily plays an important role in the regulation of these enzymes. One of the key phosphorylation sites required for the activation of all PKC isoforms lies in the T-loop of the kinase domain. Recent in vitro and transfection experiments indicate that phosphorylation of this residue can be mediated by the 3-phosphoinositide-dependent protein kinase-1 (PDK1). In this study, we demonstrate that in embryonic stem (ES) cells lacking PDK1 (PDK1-/- cells), the intracellular levels of endogenously expressed PKCalpha, PKCbetaI, PKCgamma, PKCdelta, PKCepsilon, and PKC-related kinase-1 (PRK1) are vastly reduced compared to control ES cells (PDK1+/+ cells). The levels of PKCzeta and PRK2 protein are only moderately reduced in the PDK1-/- ES cells. We demonstrate that in contrast to PKCzeta expressed PDK1+/+ ES cells, PKCzeta in ES cells lacking PDK1 is not phosphorylated at its T-loop residue. This provides the first genetic evidence that PKCzeta is a physiological substrate for PDK1. In contrast, PRK2 is still partially phosphorylated at its T-loop in PDK1-/- cells, indicating the existence of a PDK1-independent mechanism for the phosphorylation of PRK2 at this residue.
Balendran
A
A
MRC Protein Phosphorylation, MSI/WTB complex, University of Dundee, Dow Street, DD1 5EH, Dundee, UK.
Hare
G R
GR
Kieloch
A
A
Williams
M R
MR
Alessi
D R
DR
D064413:3-Phosphoinositide-Dependent Protein Kinases; D000818:Animals; D002460:Cell Line; D002478:Cells, Cultured; D004622:Embryo, Mammalian; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000737:chemistry / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D013234:Stem Cells / Q000201:enzymology; D000099221:Protein Kinase C zeta
D064413:3-Phosphoinositide-Dependent Protein Kinases; D007527:Isoenzymes; D011493:Protein Kinase C; D017346:Protein Serine-Threonine Kinases; D000099221:Protein Kinase C zeta; C061570:protein kinase N; C578562:PDPK1 protein, human