Methods in Molecular Biology TM Methods in Molecular Biology TM VOLUME 233 Edited by Alexandra C. Newton Protein Kinase C Protocols Edited by Alexandra C. Newton Protein Kinase C Protocols Ins and Outs of Protein Kinase C 3 3 From: Methods in Molecular Biology, vol. 233: Protein Kinase C Protocols Edited by: A. C. Newton © Humana Press Inc., Totowa, NJ 1 The Ins and Outs of Protein Kinase C Alexandra C. Newton 1. Protein Kinase C The seminal discovery of protein kinase C (PKC) by Nishizuka and co-workers (1) in the late 1970s provided the fi rst chapter in the story of one of the most studied enzymes in biology. The subsequent fi nding that PKC transduces signals that cause lipid hydrolysis, followed shortly thereafter by the discovery that PKC was the long sought-after receptor for the potent tumor-promoting phorbol esters, catapulted PKC to the forefront of research on signal transduc- tion. More than 35,000 research articles have been published on PKC. An abundance of reviews describe the structure, regulation, and biological function of PKC and the interested reader is referred to these for a more detailed overview than the brief one provided below (1–11). The goal of this volume is to present a lab manual on classic and novel techniques that are currently used for studying PKC. 2. PKC Primer PKC comprises a family of serine/threonine kinases whose members play a pivotal role in cell signaling. Specifi cally, PKC isozymes transduce the myriad of signals that produce lipid second messengers. Examples of physiological stimuli that activate PKC are mitogens, which operate through tyrosine kinase receptors, or catecholamines, which operate through G protein-coupled recep- tors. PKC can also be hyperactivated by treating cells with phorbol esters (see Chapter 34). These molecules bind PKC over two orders of magnitude more tightly than diacylglycerol, and coupled with their resistance to metabolism, cause essentially constitutive activation of PKC. 4 Newton There are three subclasses of PKCs, which have in common an amino- terminal regulatory domain linked to a carboxyl-terminal kinase domain (Fig. 1). The regulatory domain comprises at least one membrane-targeting module that directs PKC to the membrane in response to generation of lipid second messengers (see Chapter 8). Engaging these modules on the membrane activates PKC by providing the energy to release an autoinhibitory pseudosubstrate sequence from the substrate-binding cavity in the kinase domain, thus activating the enzyme (see Chapter 5). Conventional (α, βI, βII, γ) and novel (δ, ε, η/L, θ) PKCs are allosterically regulated by diacylglycerol, which binds to the C1 domain (this domain is a tandem repeat in most isozymes; see Fig. 1). Conventional isozymes are under additional control by Ca 2+ , which binds to the C2 domain and promotes its interaction with anionic phospholipids. Although novel PKCs contain this domain, the Ca 2+ binding pocket lacks essential aspartates involved in coordinating Ca 2+ and thus does not bind Ca 2+ . Atypical PKCs (ζ, ιλ) contain a single membrane-targeting module, the C1 domain, but the ligand-binding pocket is compromised so that it is unable to bind diacylglycerol. The function of this domain in atypical PKCs is not known. In addition to second messenger regulation, the activity of all isozymes of PKC is stimulated by phosphatidylserine: this lipid participates in anchoring the C1 domain to the membrane. The structures of the isolated C1 and C2 domains have been solved; that of the kinase domain has been modeled based on the crystal structure of the related protein kinase A (see Chapter 23). Before PKC is competent to respond to second messengers, it must fi rst be phosphorylated at three conserved positions in the kinase core (see Chap- ter 13). The fi rst phosphorylation is catalyzed by the recently discovered phosphoinositide-dependent kinase, PDK-1 (12). For conventional and novel PKCs, this phosphorylation is constitutive and part of the maturation process of the enzyme. It serves to correctly position residues in the active site for catalysis without directly activating the enzyme. Activation requires removal of the pseudosubstrate from the active site, which depends on the second messenger-mediated membrane translocation. In contrast, the phosphorylation of atypical PKCs is under moderate regulation by 3′ phosphoinositides. These isozymes do not appear to be allosterically regulated by second messengers, and it may be that phosphorylation by PDK-1 serves as the direct on/off switch for enzymatic activity. The function of PKC is exquisitely sensitive to its subcellular location (see Chapter 26). This location is not only dictated by the protein:lipid interactions described above but also by protein:protein interactions. A variety of anchoring proteins for PKC have been described. These proteins tether both inactive and active PKC at specific intracellular locations. A striking example of the importance of such scaffold proteins is in the Drosophila phototransductive Ins and Outs of Protein Kinase C 5 Fig. 1. Schematic showing the domain structure of the conventional, novel, and atypical subclasses of PKC. Indicated are the pseudosubstrate, C1 and C2 domains in the regulatory moiety, and the carboxyl-terminal kinase domain. Note that the kinase domain is sometimes subdivided into C3 and C4 domains representing the ATP-binding and substrate-binding lobes of the kinase (e.g., see Fig. 3 in Chapter 32). Adapted from ref. 9. Fig. 2. Model illustrating how the function and subcellular location of protein kinase C is under the coordinated regulation of 1. phosphorylation mechanisms, 2. cofactor binding, 3. activation-dependent dephosphorylation mechanisms, and 4. binding to scaffold proteins. Pink circles represent phosphorylation sites. The upstream kinase, PDK-1, is shown in purple. Shaded green boxes represent scaffold proteins for the various activation states of protein kinase C. Note that engaging the C2 (yellow) and C1 (orange) domains on the membrane locks PKC in the active conformation. In this conformation, the pseudosubstrate (green rectangle) is removed from the substrate- binding cavity in the kinase domain (blue circle) allowing substrate phosphorylation and down-stream signaling. 6 Newton cascade. In this system, components of the signaling cascade are coordinated on a single scaffold, the inaD protein. Disruption of the interaction of PKC with the scaffold abolishes signaling through this pathway. Once activated, PKC phosphorylates an abundance of downstream substrate proteins that regulate many distinct cellular processes (see Chapter 20). It should be noted, however, that despite two decades of research on PKC, a unifying signaling mechanism centered on PKC has remained elusive. Nonetheless, some themes are emerging. For example, various PKC family members, in particular PKCε and PKCζ have been shown to regulate cell growth and gene transcription by activating the mitogen-activated protein kinase pathway. Similarly, PKC has been implicated in cellular differentia- tion, such as neurite extension in the rat pheochromocytoma cell line PC12. Activated PKC is very sensitive to dephosphorylation and prolonged activation results in dephosphorylation and eventual proteolysis, a process referred to as downregulation. The study of PKC has been greatly helped by the use of pharmacological probes, most notably phorbol esters (see Chapter 32). Genetic approaches have also provided much insight into the function of PKC (see Chapter 36). References 1. Nishizuka, Y. (1986) Studies and perspectives of protein kinase C. Science 233, 305–312. 2. Blumberg, P. M., Acs, G., Areces, L. B., Kazanietz, M. G., Lewin, N. E., Szallasi. Z. (1994) Protein kinase C in signal transduction and carcinogenesis. Prog. Clin. Biol. Res. 387, 3–19. 3. Nishizuka, Y. (1995) Protein kinase C and lipid signaling for sustained cellular responses. FASEB J. 9, 484–496. 4. Mellor, H. and Parker, P. J. (1998) The extended protein kinase C superfamily. Biochem. J. 332, 281–292. 5. Toker, A. (1998) Signaling through protein kinase C. Front. Biosci. 3, D1134–D1147. 6. Jaken, S. and Parker P. J. (2000) Protein kinase C binding partners. Bioessays 22, 245–254. 7. Parekh, D. B., Ziegler, W., and Parker P. J. (2000) Multiple pathways control protein kinase C phosphorylation. EMBO J. 19, 496–503. 8. Mochly-Rosen, D. and Gordon A. S. (1998) Anchoring proteins for protein kinase C: a means for isozyme selectivity. FASEB J. 12, 35–42. 9. Newton, A.C. and Johnson J. E. (1998) Protein kinase C: a paradigm for regulation of protein function by two membrane-targeting modules. Biochim. Biophys. Acta 1376, 155–172. 10. Ron, D. and Kazanietz M. G. (1999) New insights into the regulation of protein kinase C and novel phorbol ester receptors. FASEB J. 13, 1658–1676. Ins and Outs of Protein Kinase C 7 11. Newton, A. C. (2001) Protein kinase C: structural and spatial regulation by phosphorylation, cofactors, and macromolecular interactions. Chem. Rev. 101, 2353–2364. 12. Toker, A. and Newton A. (2000) Cellular signalling: pivoting around PDK-1. Cell 103, 185–188. 8 Newton Early Studies of Protein Kinase C 9 9 From: Methods in Molecular Biology, vol. 233: Protein Kinase C Protocols Edited by: A. C. Newton © Humana Press Inc., Totowa, NJ 2 Early Studies of Protein Kinase C A Historical Perspective Yasutomi Nishizuka and Ushio Kikkawa 1. Introduction The covalent attachment of phosphate to either seryl or threonyl residues of proteins was identifi ed fi rst by F. Lipmann and P. A. Levene at the Rockefeller Institute for Medical Research (Rockefeller University, New York) in 1932; these researchers were interested in the chemical nature of acidic macromol- ecules present in the cell nucleus (paranucleic acid was the term used by Levene). This nuclear material was presumably a mixture of what we now call transcription factors. The enzyme responsible for this protein modifi cation, casein kinase (phosvitin kinase), was subsequently found by M. Rabinowitz and Lipmann, but no obvious function was assigned for this enzyme. Another line of study focusing on glycogen metabolism initiated by C. F. Cori and G. T. Cori at St. Louis in the early 1940s, and by eminent investigators, such as E. W. Sutherland, T. W. Rall, E. G. Krebs, E. Fischer, and J. Larner, clarifi ed the role of reversible phosphorylation in controlling the breakdown and resynthesis of glycogen. In the mid-1960s, Y. Nishizuka spent 1 year as an NIH International Postdoctoral Research Fellow in the laboratory of Lipmann to work on the elongation factors of protein synthesis in Escherichia coli. Discussions there about a possible relationship between nuclear phosphoproteins and bacterial adaptive enzymes induced by cyclic AMP sparked Nishizuka’s lifelong interest in protein kinases in hormone actions. At the end of the 1960s, when Nishizuka moved to Kobe, Krebs and his colleagues announced that cyclic AMP activates glycogen phosphorylase kinase kinase, known as protein kinase A (PKA) today 10 Nishizuka and Kikkawa (1). H. Yamamura and Nishizuka at that time in Kobe isolated a functionally unidentifi ed kinase from rat liver with histone as phosphate acceptor and confi rmed that cyclic AMP greatly stimulated its catalytic activity. Soon, in 1970, four laboratories (Krebs, Lipmann, G. N. Gill, and Yamamura and Nishizuka) concurrently reported that PKA consists of catalytic and regulatory subunits and that cyclic AMP activates the enzyme by dissociating these subunits (Fig. 1). The 1970s marked the initiation of several important studies of protein kinases (Table 1). In the case of cyclic GMP-dependent protein kinase (PKG), discovered by J. F. Kuo and P. Greengard in the brain in 1970, M. Inoue in the Kobe group found that this enzyme, unlike PKA, is a single polypeptide chain and is activated by cyclic GMP, which binds simply to its regulatory region to promote catalytic activity. They found that a constitutively active enzyme frag- ment insensitive to the cyclic nucleotide could be generated by limited prote- olysis (2). This enabled us to fi nd a new enzyme, Ca 2+ -activated, phospholipid- dependent protein kinase that is protein kinase C (PKC). 2. PKC and Link to Receptor Higher levels of an active fragment named protein kinase M (PKM; M for its only known requirement, Mg 2+ ), which was assumed to be derived from PKG, were found in previously frozen rat brain compared with freshly obtained brain, where not much fragment was detected. Freezing and thawing resulted in Fig. 1. Mode of activation of three protein kinases. Early Studies of Protein Kinase C 11 the appearance of the active fragment, suggesting the presence of a proenzyme susceptible to limited proteolysis, perhaps by a Ca 2+ -dependent protease, later called calpain (3). We soon noticed the existence of such a putative proenzyme in the brain; curiously, it was not sensitive to any cyclic nucleotide, but produced PKM upon limited proteolysis. Attempts to identify the protease responsible for this proteolysis led us to uncover large quantities of an activating substance associated with the membrane. It was not a protease, but simply anionic phospholipids, particularly phosphatidylserine. Even more curiously, crude phospholipids extracted from brain membranes could support activation of the enzyme in the absence of added Ca 2+ , whereas pure phospholipids obtained from erythrocyte membranes could not produce any enzyme activation unless a higher concentration of Ca 2+ was added to the reaction mixture (Fig. 2). Analysis of the lipid impurities on a silicic acid column led us to conclude that diacylglycerol was an essential activator (4). This observation suggested a critical link between protein phosphorylation and the signal-induced hydrolysis of inositol phospholipids that was described by M. R. Hokin and L. E. Hokin with acetylcholine-stimulated pancreatic acinar cells in the early 1950s (5). A ubiquitous distribution of PKC in mammalian and other animal tissues was immediately confi rmed by J. F. Kuo in Atlanta. Today, we know that the enzyme is distributed more widely, and it is also extensively studied for its role in yeast, Nematoda, and the fl y. Table 1 Discovery of Protein Kinases (1955–1980) Years Researchers Studies 1955 E. Fischer and E. G. Krebs Glycogen phosphorylase kinase 1960 M. Rabinowitz and F. Lipmann Casein kinase (phosvitin kinase) 1963 J. Larner Glycogen synthetase kinase 1964 T. Langan and F. Lipmann Histone kinase 1968 D. A. Walsh, J. P. Parkins, Protein kinase A and E. G. Krebs 1970 J. F. Kuo and P. Greengard Protein kinase G 1977 P. Greengard Calmodulin-dependent protein kinase K. Yagi; D. G. Hartshorne Myosin light chain kinase P. Cohen Calmodulin in phosphorylase kinase Y. Nishizuka and colleagues Protein kinase C 1978 R. L. Erikson Src protein kinase 1979 H. Hunter Protein tyrosine kinase 1980 S. Cohen Receptor tyrosine kinase (epidermal growth factor receptor) [...]... amplification (cycle profile: 94 C/ 5 min; 10 × 94 C/ 15 sec, 56 C/ 30 sec, 72 C/ 2 min; 15 × 94 C/ 15 sec, 56 C/ 30 sec, 72 C/ 2 min, plus cycle elongation of 20 sec for each cycle; 72 C/ 7 min), a rat PKCδ full-length cDNA clone is used as template The resulting cDNA of 2048 bp is cut with NdeI and EcoRI and cloned into the NdeI-EcoRI-cut expression vector pET28 The resulting plasmid coding for PKCδ with an... the Vector pET28 For the construction of a PKCδ full-length cDNA with an NdeI restriction site at the initiation signal ATG and an EcoRI restriction site behind the stop codon TGA, the following oligonucleotide primers are used: 5′-AAA GGA TCC CAT ATG GCA CCG TTC CTG CGC-3′ as the 5′-primer and 5′-TCT GGG AAT TCA CTA CTA TTC CAG GAA TTG CTC-3′ as the 3′-primer For polymerase chain reaction amplification... types of technology: 1) recombination in Saccharomyces cerevisiae (5); 2) enzymatic in vitro recombination using Cre-lox (6); 3) direct cloning at a unique restriction site (7) or speci c vector-compatible overhangs (8); and 4) site-speci c transposition in Escherichia coli (9) (Types 3 and 4 are now commercially available; Novagen’s pBac-LIC and Invitrogen’s Bac-to-Bac, respectively.) These technologies... sequence Expression and Purification of PKC from Insect Cells 31 4 Novagen’s BacVector triple cut viral DNA contains a lacZ gene that is replaced after rescue by the plasmid containing the foreign gene, all recombinants will produce colorless plaques when stained with X-gal You can increase the recombination efficiency further by using this selection system when a pAcGHLT transfer vector is co-transfected... Kikkawa Fig 2 Activation of PKC by phospholipids, diacylglycerol, and Ca2+ To obtain evidence that diacylglycerol is the intracellular mediator of hormone actions, we needed a method to activate PKC within intact cells Diacylglycerols that have two long chain fatty acids could not be readily intercalated into the cell membrane If, however, one of the fatty acids is replaced with a short chain, acetyl group,... phosphatidylinositol 3 -kinase activation, which was described first by L Cantley in Boston 10 years ago (24) Another cross-talk for PKC activation through tyrosine phosphorylation of the enzyme molecule is becoming clearer (25) The catalytic and functional competence of the isoforms appears also to depend on the speci c intracellular localization of each, namely their translocation or targeting to particular compartments,... which helps isolate the highly purified active recombinant enzyme easily and quickly The baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV) has become a popular vehicle for the cloning and expression of recombinant proteins in insect cells The transcriptional activity in the very late phase of insect cells infected with AcNPV is dedicated to the polyhedrin and p10 promoters, which... Maintenance of Sf9 Cells 1 2 3 4 5 6 7 EX-CELL 420 medium (JRH Bioscience) Fetal bovine serum (Gibco) Antibiotic-antimycotic (×100, Gibco) 28 C incubator 60-mm, 100-mm culture dishes Erlenmeyer flask (500 mL) Orbital incubator shaker (Taitek) 2.2 Virus DNA and Transfer Vectors 1 2 3 4 5 6 BacVector 3000 triple cut virus DNA (Novagen) pBlueBacHis (Invitrogen) pGEX4T-1 (Pharmacia) pAcGHLT (Pharmingen) cDNA clones... However, pBlueBacHis or pBlueBacHisGST(pR538) transfer vector contains 5′ lacZ fragment, so even the recombinant plaques produced are blue In both cases, staining by X-gal is usually not necessary because of the high recombination efficiency using the triple cut viral DNA 5 It is not recommended that the virus stocks are consecutively passaged many times as this will lead to the occurrence and accumulation... recombinant baculovirus expression vectors at high frequency BioTechniques 14, 810–817 5 Patel, G., Nasmyth, K., and Jones, N (1992) A new method for the isolation of recombinant baculovirus Nucleic Acids Res 20, 97–104 6 Peakman, T C. , Harris, R A., and Gewert, D R (1992) Highly efficient generation of recombinant baculoviruses by enzymatically medicated site-speci c in vitro recombination Nucleic Acids . technology: 1) recombination in Saccharomyces cerevisiae (5); 2) enzymatic in vitro recombination using Cre-lox (6); 3) direct cloning at a unique restriction site (7) or specifi c vector-compatible. the occasion of the Fourth International Conference on Cyclic Nucleotides, Nishizuka had an exciting time discussing with M. Castagna in Villejuif a potential connection between PKC activation. Molecular Biology TM Methods in Molecular Biology TM VOLUME 233 Edited by Alexandra C. Newton Protein Kinase C Protocols Edited by Alexandra C. Newton Protein Kinase C Protocols Ins and Outs of Protein