Joanne E. Johnson

Regulation of protein kinase C ζ by PI 3-kinase and PDK-1

BACKGROUND Protein kinase C zeta (PKC zeta) is a member of the PKC family of enzymes and is involved in a wide range of physiological processes including mitogenesis, protein synthesis, cell survival and transcriptional regulation. PKC zeta has received considerable attention recently as a target of phosphoinositide 3-kinase (PI 3-kinase), although the mechanism of PKC zeta activation is, as yet, unknown. Recent reports have also shown that the phosphoinositide-dependent protein kinase-1 (PDK-1), which binds with high affinity to the PI 3-kinase lipid product phosphatidylinositol-3,4,5-trisphosphate (Ptdins-3,4,5-P3), phosphorylates and potently activates two other PI 3-kinase targets, the protein kinases Akt/PKB and p70S6K. We therefore investigated whether PDK-1 is the kinase that activates PKC zeta. RESULTS In vivo, PI 3-kinase is both necessary and sufficient to activate PKC zeta. PDK-1 phosphorylates and activates PKC zeta in vivo, and we have shown that this is due to phosphorylation of threonine 410 in the PKC zeta activation loop. In vitro, PDK-1 phosphorylates and activates PKC zeta in a Ptdins-3,4,5-P3-enhanced manner. PKC zeta and PDK-1 are associated in vivo, and membrane targeting of PKC zeta renders it constitutively active in cells. CONCLUSIONS Our results have identified PDK-1 as the kinase that phosphorylates and activates PKC zeta in the PI 3-kinase signaling pathway. This phosphorylation and activation of PKC zeta by PDK-1 is enhanced in the presence of Ptdins-3,4-5-P3. Consistent with the notion that PKCs are enzymes that are regulated at the plasma membrane, a membrane-targeted PKC zeta is constitutively active in the absence of agonist stimulation. The association between PKC zeta and PDK-1 reveals extensive cross-talk between enzymes in the PI 3-kinase signaling pathway.

Structure of the membrane binding domain of CTP:phosphocholine cytidylyltransferase.

It has been proposed that the domain of the regulatory enzyme, CTP:phosphocholine cytidylyltransferase, which mediates reversible binding of the enzyme to membranes, is an amphipathic alpha-helix of approximately 60 amino acid residues and that this domain is adjacent to the putative active site domain of this enzyme. Circular dichroism indicated that the secondary structures of two overlapping peptides spanning this region were predominantly alpha-helical in the presence of PG vesicles or sodium dodecyl sulfate micelles. Interproton distances were obtained from two-dimensional NMR spectroscopic measurements to solve the structures of these two peptides. The C-terminal 22 amino acid peptide segment (corresponding to Val267-Ser288) was a well-defined alpha-helix over its length. The N-terminal 33-mer (corresponding to Asn236-Glu268) was composed of an alpha-helix from Glu243 to Lys266, a well-structured bend of about 50 degrees at Tyr240-His241-Leu242, and an N-terminal four-residue helix. It is proposed that the three residues involved in generating the bend act as the hinge between the catalytic and regulatory domains. The nonpolar faces of the 33-mer and 22-mer were interrupted by Ser260, Ser271, and Ser282. These residues may serve to limit the hydrophobicity and facilitate reversible and lipid-selective membrane binding. The hydrophobic faces of the helices were flanked by a set of basic amino acid residues on one side and basic amino acid residues interspersed with glutamates on the other. The disposition of these side chains gives clues to the basis for the specificities of these peptides for anionic surfaces.

Protected-site phosphorylation of protein kinase C in hippocampal long- term potentiation

Abstract: One important aspect of synaptic plasticity is that transient stimulation of neuronal cell surface receptors can lead to long‐lasting biochemical and physiological effects in neurons. In long‐term potentiation (LTP), generation of autonomously active protein kinase C (PKC) is one biochemical effect persisting beyond the NMDA receptor activation that triggers plasticity. We previously observed that the expression of early LTP is associated with a phosphatase‐reversible alteration in PKC immunoreactivity, suggesting that autophosphorylation of PKC might be elevated in LTP. In the present studies we tested the hypothesis that PKC phosphorylation is persistently increased in the early maintenance of LTP. We generated an antiserum that selectively recognizes the α and βII isoforms of PKC autophosphorylated in the C‐terminal domain. Using western blotting with this antiserum we observed an NMDA receptor‐mediated increase in phosphorylation of PKC 1 h after LTP was induced. How is the increased phosphorylation maintained in the cell in the face of ongoing phosphatase activity? We observed that dephosphorylation of PKC in vitro requires the presence of cofactors normally serving to activate PKC, i.e., Ca2+, phosphatidylserine, and diacylglycerol. Based on these observations and computer modeling of the three‐dimensional structure of the PKC catalytic core, we propose a “protected site” model of PKC autophosphorylation, whereby the conformation of PKC regulates accessibility of the phosphates to phosphatase. Although we have proposed the protected site model based on our studies of PKC phosphorylation in LTP, phosphorylation of protected sites might be a general biochemical mechanism for the generation of stable, long‐lasting physiologic changes.

Crystal Structure of a Mammalian CTP: Phosphocholine Cytidylyltransferase Catalytic Domain Reveals Novel Active Site Residues within a Highly Conserved Nucleotidyltransferase Fold

CTP:phosphocholine cytidylyltransferase (CCT) is the key regulatory enzyme in the synthesis of phosphatidylcholine, the most abundant phospholipid in eukaryotic cell membranes. The CCT-catalyzed transfer of a cytidylyl group from CTP to phosphocholine to form CDP-choline is regulated by a membrane lipid-dependent mechanism imparted by its C-terminal membrane binding domain. We present the first analysis of a crystal structure of a eukaryotic CCT. A deletion construct of rat CCTα spanning residues 1–236 (CCT236) lacks the regulatory domain and as a result displays constitutive activity. The 2.2-Å structure reveals a CCT236 homodimer in complex with the reaction product, CDP-choline. Each chain is composed of a complete catalytic domain with an intimately associated N-terminal extension, which together with the catalytic domain contributes to the dimer interface. Although the CCT236 structure reveals elements involved in binding cytidine that are conserved with other members of the cytidylyltransferase superfamily, it also features nonconserved active site residues, His-168 and Tyr-173, that make key interactions with the β-phosphate of CDP-choline. Mutagenesis and kinetic analyses confirmed their role in phosphocholine binding and catalysis. These results demonstrate structural and mechanistic differences in a broadly conserved protein fold across the cytidylyltransferase family. Comparison of the CCT236 structure with those of other nucleotidyltransferases provides evidence for substrate-induced active site loop movements and a disorder-to-order transition of a loop element in the catalytic mechanism.

A Putative Phosphatidylserine Binding Motif Is Not Involved in the Lipid Regulation of Protein Kinase C

Protein kinase C is specifically regulated by diacylglycerol and the amino phospholipid, phosphatidylserine. The molecular basis for the phosphatidylserine specificity was recently proposed to arise from the presence of a putative phosphatidylserine binding motif, FXFXLKXXXKXR, localized in the C2 domain of protein kinase C (Igarashi, K., Kaneda, M., Yamaji, A., Saido, T. C., Kikkawa, U., Ono, U., Inoue, K., and Umeda, M. (1995) J. Biol. Chem. 270, 29075–29078). To determine whether this motif mediates the interaction of protein kinase C with phosphatidylserine, the carboxyl-terminal basic residues were mutated to Ala in protein kinase C βII (K236A and R238A), and the phosphatidylserine regulation of the mutant enzyme was examined. Membrane binding and activity measurements revealed that the phosphatidylserine regulation for the mutant protein was indistinguishable from that of wild-type protein kinase C. Specifically, neither the apparent membrane affinity for phosphatidylserine-containing membranes in the presence or absence of diacylglycerol nor the phosphatidylserine-dependence for activation was affected by removal of the conserved basic residues at the carboxyl terminus of the consensus sequence. In addition, a synthetic peptide corresponding to the amino terminus of the consensus sequence (FTFNVK) had no effect on the concentration of phosphatidylserine resulting in half-maximal activation of protein kinase C. These results reveal that the carboxyl-terminal basic residues in the consensus motif FXFXLKXXXKXR are not responsible for the phosphatidylserine selectivity of protein kinase C and that, furthermore, the region of the C2 domain containing this motif is not involved in the membrane binding of protein kinase C.