Identification of Tyrosine Phosphorylation Sites on 3-Phosphoinositide-dependent Protein Kinase-1 and Their Role in Regulating Kinase Activity

Abstract

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 tyrosinephosphorylated 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 ϳ6-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.3-Phosphoinositide-dependent protein kinase-1 (PDK1) 1 appears to play a central regulatory role in many cell-signaling pathways (1-12). Several substrates of PDK1 have so far been identified, including protein kinase B (PKB), p70 ribosomal protein S6 kinase (p70S6K), cyclic AMP-dependent protein kinase (PKA), protein kinase C (PKC), serum and glucocorticoidinducible kinase (SGK), p90 ribosomal protein S6 kinase (RSK), and p21-activated kinase-1 (1, 2, 9, 13-31). However, most of these putative PDK1 substrates were identified using in vitro phosphorylation experiments, and not all the substrate proteins appear to be physiological targets for PDK1 in vivo. Experiments with embryonic stem (ES) cells ablated for both alleles of PDK1 revealed that PKB, p70S6K, and RSK are no longer phosphorylated or activated by agonists that are potent stimuli in normal ES cells, providing in vivo evidence that these three enzymes are dependent on PDK1 for activation (32). The regulation of the other potential substrates of PDK1 is apparently unaffected in the knock-out cell line (32). PKB, p70S6K, RSK, and SGK, members of the AGC family of protein kinases, are apparently maintained in the cytoplasm in an inactive state. Upon stimulation of receptor tyrosine kinases, phosphoinositide 3-kinase (PI3K) becomes activated, generating the phospholipid second messengers, PtdIns(3,4,5)P 3 and PtdIns(3,4)P 2 . These lipids then mediate the phosphorylation and activation of PDK1 targets through diverse mechanisms (...

Increased cytoplasmic levels of CIS, SOCS1, SOCS2, or SOCS3 are required for nuclear translocation

“…Gang Min Hur at Chungnam University, Korea. The pEF-Flag-CIS, pEF-Flag-SOCS1, pEF-Flag-SOCS2, pEF-Flag-SOCS3, PKBa, and Stat3-C plasmids have been described previously [16][17][18].…”

Analysis of 3-phosphoinositide-dependent kinase-1 signaling and function in ES cells

“…Although initially thought to be a constitutively active kinase, more recent evidence suggests that its activity might be regulated by phosphorylation under some circumstances [12,13].…”

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

“…PDK1s are commonly phosphorylated at several sites, some of which are important for enzyme activity, others for substrate association as well as for binding of regulatory molecules such as 14-3-3 proteins [14][15][16]22]. We aimed at identifying phosphorylation sites in AtPDK1 and their participation in either its activity, or in the interaction and/or phosphorylation of AtS6k2.…”

“…Mammalian PDK1 undergoes autophosphorylation via both cis (intramolecular) and trans (intermolecular) mechanisms, and several autophosphorylation sites have been identified [12][13][14][15]. Autophosphorylation of Ser-241 in human PDK1 is essential for its activity and the corresponding residue in mouse PDK1 was recently shown to be autophosphorylated via a trans mechanism dependent on PDK1 dimerisation [12,13].…”

“…Apart from PDK1 itself, no serine/threonine kinases able to phosphorylate PDK1 have been identified. 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.…”