Dual role of pseudosubstrate in the coordinated regulation of protein kinase C by phosphorylation and diacylglycerol.

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 betaII 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.

Sphingosine is a novel activator of 3-phosphoinositide-dependent kinase 1.

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.

Regulation of conventional protein kinase C isozymes by phosphoinositide-dependent kinase 1 (PDK-1).

BACKGROUND: Phosphorylation critically regulates the catalytic function of most members of the protein kinase superfamily. One such member, protein kinase C (PKC), contains two phosphorylation switches: a site on the activation loop that is phosphorylated by another kinase, and two autophosphorylation sites in the carboxyl terminus. For conventional PKC isozymes, the mature enzyme, which is present in the detergent-soluble fraction of cells, is quantitatively phosphorylated at the carboxy-terminal sites but only partially phosphorylated on the activation loop. RESULTS: This study identifies the recently discovered phosphoinositide-dependent kinase 1, PDK-1, as a regulator of the activation loop of conventional PKC isozymes. First, studies in vivo revealed that PDK-1 controls the amount of mature (carboxy-terminally phosphorylated) conventional PKC. More specifically, co-expression of the conventional PKC isoform PKC betaII with a catalytically inactive form of PDK-1 in COS-7 cells resulted in both the accumulation of non-phosphorylated PKC and a corresponding decrease in PKC activity. Second, studies in vitro using purified proteins established that PDK-1 specifically phosphorylates the activation loop of PKC alpha and betaII. The phosphorylation of the mature PKC enzyme did not modulate its basal activity or its maximal cofactor-dependent activity. Rather, the phosphorylation of non-phosphorylated enzyme by PDK-1 triggered carboxy-terminal phosphorylation of PKC, thus providing the first step in the generation of catalytically competent (mature) enzyme. CONCLUSIONS: 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.

Inhibition of Raf-1 signaling by a monoclonal antibody, which interferes with Raf-1 activation and with Mek substrate binding.

Raf-1 is a serine/threonine specific kinase that integrates signaling by a large number of mitogens to elicit a transcriptional response in the nucleus. Activated Raf-1 phosphorylates and activates MAPK/ERK kinase Mek), thus initiating the Mek--> MAP kinase cascade, which ultimately results in the phosphorylation and activation of transcription factors by MAP kinase. Here we have characterized the mechanism by which monoclonal antibody URP26K, which binds to an epitope in the Raf-1 kinase domain, inhibits intracellular signal transduction. This antibody preferentially immunoprecipitated the underphosphorylated, non-activated form of Raf-1 from quiescent cells. Baculovirus-expressed Raf-1 immunoprecipitated with URP26K was largely refractory to phosphorylation and activation mediated by protein kinase C (PKC)alpha or the tyrosine kinase Lck. In addition, URP26K reduced the binding of Raf-1 to its substrate Mek in vitro, but did not disturb the association of Raf-1 with Ras. Microinjection of URP26K into Rat-1 cells blocked DNA synthesis initiated by serum, insulin and various purified growth factors, but it did not block DNA synthesis initiated by v-ras. Microinjected URP26K also impaired the expression of stably transfected beta-galactosidase reporter genes regulated by minimal promoter elements. These results demonstrate, (i) that the URP26K monoclonal antibody inhibits Raf-1 by preventing activating Raf-1 phosphorylation and/or association with its substrate Mek, (ii) that inhibition of Raf-1 by URP26K does not interfere with Ras-induced DNA synthesis. In contrast to dominant negative Raf-1 mutants, which also block Ras signaling by binding to the Ras effector domain, antibody mediated Raf-1 inhibition thus reveals a branchpoint of mitogenic signaling at the level of Ras.

Protein kinase C is regulated in vivo by three functionally distinct phosphorylations.

BACKGROUND: Protein kinase Cs are a family of enzymes that transduce the plethora of signals promoting lipid hydrolysis. Here, we show that protein kinase C must first be processed by three distinct phosphorylations before it is competent to respond to second messengers. RESULTS: We have identified the positions and functions of the in vivo phosphorylation sites of protein kinase C by mass spectrometry and peptide sequencing of native and phosphatase-treated kinase from the detergent-soluble fraction of cells. Specifically, the threonine at position 500 (T500) on the activation loop, and T641 and S660 on the carboxyl terminus of protein kinase C beta II are phosphorylated in vivo. T500 and S660 are selectively dephosphorylated in vitro by protein phosphatase 2A to yield an enzyme that is still capable of lipid-dependent activation, whereas all three residues are dephosphorylated by protein phosphatase 1 to yield an inactive enzyme. Biochemical analysis reveals that protein kinase C autophosphorylates on S660, that autophosphorylation on S660 follows T641 autophosphorylation, that autophosphorylation on S660 is accompanied by the release of protein kinase C into the cytosol, and that T500 is not an autophosphorylation site. CONCLUSIONS: 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.

In vivo regulation of protein kinase C by trans-phosphorylation followed by autophosphorylation.

Dephosphorylation by the catalytic subunits of protein phosphatases 1 (CS1) and 2A (CS2) reveals that mature protein kinase C is phosphorylated at two distinct sites. Treatment of protein kinase C beta II with CS1 causes a significant increase in the protein's electrophoretic mobility (approximately 4 kDa) and a coincident loss in catalytic activity. The CS1-dephosphorylated enzyme cannot autophosphorylate or be phosphorylated by mature protein kinase C, indicating that a different kinase catalyzes the phosphorylation at this site. The loss of activity is consistent with dephosphorylation on protein kinase C's activation loop (Orr, J. W., and Newton, A. C., (1994) J. Biol. Chem. 269, 27715-27718). Treatment with CS2 results in a smaller shift in electrophoretic mobility (approximately 2 kDa) and no loss in catalytic activity. Furthermore, the CS2-dephosphorylated form can autophosphorylate and thus regain the electrophoretic mobility of mature enzyme, consistent with dephosphorylation at protein kinase C's carboxyl-terminal autophosphorylation site, which is modified in vivo (Flint, A. J., Paladini, R. D., and Koshland, D. E., Jr. (1990) Science 249, 408-411). In summary, two phosphorylations process protein kinase C to generate the mature form: a transphosphorylation that renders the kinase catalytically competent and an autophosphorylation that may be important for the subcellular localization of the enzyme.