Identification of tyrosine phosphorylation sites on 3-phosphoinositide-dependent protein kinase-1 and their role in regulating kinase activity.
3-Phosphoinositide-dependent protein kinase-1 (PDK1) plays a central role in signal transduction pathways that activate phosphoinositide 3-kinase. Despite its key role as an upstream activator of enzymes such as protein kinase B and p70 ribosomal protein S6 kinase, the regulatory mechanisms controlling PDK1 activity are poorly understood. PDK1 has been reported to be constitutively active in resting cells and not further activated by growth factor stimulation (Casamayor, A., Morrice, N. A., and Alessi, D. R. (1999) Biochem. J. 342, 287-292). Here, we report that PDK1 becomes tyrosine-phosphorylated and translocates to the plasma membrane in response to pervanadate and insulin. Following pervanadate treatment, PDK1 kinase activity increased 1.5- to 3-fold whereas the activity of PDK1 associated with the plasma membrane increased approximately 6-fold. The activity of PDK1 localized to the plasma membrane was also increased by insulin treatment. Three tyrosine phosphorylation sites of PDK1 (Tyr-9 and Tyr-373/376) were identified using in vivo labeling and mass spectrometry. Using site-directed mutants, we show that, although phosphorylation on Tyr-373/376 is important for PDK1 activity, phosphorylation on Tyr-9 has no effect on the activity of the kinase. Both of these residues can be phosphorylated by v-Src tyrosine kinase in vitro, and co-expression of v-Src leads to tyrosine phosphorylation and activation of PDK1. Thus, these data suggest that PDK1 activity is regulated by reversible phosphorylation, possibly by a member of the Src kinase family.
Park
J
J
Friedrich Miescher Institute, Maulbeerstrasse 66, Basel CH-4058, Switzerland.
Hill
M M
MM
Hess
D
D
Brazil
D P
DP
Hofsteenge
J
J
Hemmings
B A
BA
D064413:3-Phosphoinositide-Dependent Protein Kinases; D000595:Amino Acid Sequence; D001692:Biological Transport; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D002478:Cells, Cultured; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism*; D014638:Vanadates / Q000494:pharmacology; D019061:src-Family Kinases / Q000378:metabolism
D004791:Enzyme Inhibitors; C076715:pervanadate; D014638:Vanadates; D014443:Tyrosine; D019061:src-Family Kinases; D064413:3-Phosphoinositide-Dependent Protein Kinases; C578562:PDPK1 protein, human; D017346:Protein Serine-Threonine Kinases