Structural and biochemical analyses of native and phosphatase-treated protein kinase C indicate that protein kinase C is processed by three phosphorylations. Firstly, trans-phosphorylation on the activation loop (T500) renders it catalytically competent to autophosphorylate. Secondly, a subsequent autophosphorylation on the carboxyl terminus (T641) maintains catalytic competence. Thirdly, a second autophosphorylation on the carboxyl terminus (S660) regulates the enzyme's subcellular localization. The conservation of each of these residues (or an acidic residue) in conventional, novel and atypical protein kinase Cs underscores the essential role for each in regulating the protein kinase C family.

We have shown that PDK-1 controls the phosphorylation of conventional PKC isozymes in vivo. Studies performed in vitro establish that PDK-1 directly phosphorylates PKC on the activation loop, thereby allowing carboxy-terminal phosphorylation of PKC. These data suggest that phosphorylation of the activation loop by PDK-1 provides the first step in the processing of conventional PKC isozymes by phosphorylation.

The activity of protein kinase C is reversibly regulated by an autoinhibitory pseudosubstrate, which blocks the active site of the enzyme in the absence of activators. However, before it can be allosterically regulated, protein kinase C must first be processed by three ordered phosphorylations, the first of which is modification of the activation loop catalyzed by the phosphoinositide-dependent kinase-1 (PDK-1). Here we use limited proteolysis to show that 1) newly synthesized protein kinase C adopts a conformation in which its pseudosubstrate sequence is removed from the active site, and 2) this exposure is essential to allow PDK-1 to phosphorylate the enzyme. Precursor (unphosphorylated) protein kinase C ␤II obtained by 1) in vitro transcription and translation, 2) expression of a phosphorylation-deficient mutant (T500V), or 3) in vivo labeling with a pulse of [ 35 S]cysteine/methionine is cleaved at the amino-terminal pseudosubstrate by the endoproteinase Arg-C. In marked contrast to mature (phosphorylated) enzyme, proteolysis occurs in the absence of lipid activators, revealing that precursor protein kinase C has its pseudosubstrate sequence removed constitutively. Additionally, we show that PDK-1 is unable to phosphorylate protein kinase C when the active site is sterically blocked by a peptide substrate. Neither can mature enzyme be dephosphorylated when the active site is blocked by binding either the pseudosubstrate sequence or a heterologous substrate. Thus, the accessibility of the activation loop to both phosphorylation and dephosphorylation requires an exposed pseudosubstrate. In summary, newly synthesized protein kinase C adopts a conformation in which its pseudosubstrate sequence is removed from the active site, rendering the activation loop accessible to phosphorylation by PDK-1. Phosphorylation serves as a conformational switch to position the pseudosubstrate so that it blocks the active site, a conformation that is maintained until stimulus-dependent membrane binding releases it, thus activating the enzyme.The protein kinase Cs comprise a family of enzymes that are regulated by two distinct mechanisms: phosphorylation and membrane targeting (reviewed in Ref. 1). In the first regulatory mechanism, protein kinase C isozymes are rendered catalytically competent by a series of ordered phosphorylation events, the first of which has recently been shown to be catalyzed by PDK-1 1 (2-4). PDK-1 phosphorylates a segment near the entrance to the active site referred to as the activation loop; the phosphorylated position corresponds to Thr 500 in the conventional protein kinase C ␤II (5). This phosphorylation serves to correctly position residues for catalysis and triggers the autophosphorylation of two conserved positions at the carboxyl terminus, a turn motif (Thr 641 in protein kinase C ␤II), and a hydrophobic motif (Ser 660 in protein kinase C ␤II) (6). The phosphorylated "mature" species localizes to the cytosol where it is maintained in an inactive conformation by an autoinhibitory pseudosubstrate seq...

3-Phosphoinositide-dependent kinase 1 (PDK1) has previously been shown to phosphorylate the activation loop of several AGC kinase family members. In this study, we show that p21-activated kinase 1, the activity of which is regulated by the GTP-bound form of Cdc42 and Rac and by sphingosine, is phosphorylated by PDK1. Phosphorylation of p21-activated kinase 1 by PDK1 occurred only in the presence of sphingosine, which increased PDK1 autophosphorylation 25-fold. Sphingosine increased PDK1 autophosphorylation in a concentration-dependent manner and significantly increased phosphate incorporation into known PDK1 substrates. Studies on the lipid requirement for PDK1 activation found that both sphingosine isoforms and stearylamine also increased PDK1 autophosphorylation. However, C 10 -sphingosine, octylamine, and stearic acid were unable to increase PDK1 autophosphorylation, indicating that both a positive charge and a lipid tail containing at least a C 10 -carbon backbone were required for PDK1 activation. Three PDK1 autophosphorylation sites were identified after stimulation with sphingosine in a serine-rich region located between the kinase domain and the pleckstrin homology domain using two-dimensional phosphopeptide maps and matrix assisted laser desorption/ionization mass spectroscopy. Increased phosphorylation of endogenous Akt at threonine 308 was observed in COS-7 cells expressing wild type PDK1, but not catalytically inactive PDK1, when cellular sphingosine levels were elevated by treatment with sphingomyelinase. Sphingosine thus appears to be a true PDK1 activator.3-Phosphoinositide-dependent kinase 1 (PDK1) 1 is a recently described kinase containing a pleckstrin homology domain (1). The pleckstrin homology domain has been demonstrated to bind the lipid products of the phosphatidylinositol 3-OH-kinase reaction, phosphatidylinositol 3,4 bisphosphate, and phosphatidylinositol 3,4,5 trisphosphate (PtdIns 3,4,5 P 3 ), with low nanomolar affinity, and it redistributes PDK1 to the membrane without increasing PDK1 autophosphorylation or incorporation of phosphate into substrate (2). PDK1 activity has been described as constitutive and has been demonstrated to phosphorylate a conserved threonine in kinase subdomain VIII of AGC family kinase family members, including Akt, p70 S6 kinase, protein kinase A (PKA), and a variety of PKC isoforms (1, 3-7). Phosphorylation of the conserved threonine by PDK1 has proven to be a crucial step in activating these enzymes (1).p21 activated kinase 1 (PAK1) becomes active in the presence of either the GTP-bound form of the Rho family GTPases Cdc42 or Rac1, or the positively charged lipid sphingosine (8, 9). Activation of PAK1 is characterized by autophosphorylation of 7-8 serine/threonine amino acids and an increase in substrate phosphorylation (10). Although it is not an AGC kinase family member, PAK1 contains a conserved threonine in kinase subdomain VIII equivalent to the PDK1 phosphorylation site in AGC kinase family members. Although this site has been reported to be a PAK1 ...

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