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most likely s i t e . 74 The power of this approach can be further enhanced by making complementary point mutations in the protein substrate. With improvements in the potency of peptide substrates it is likely that applications of affinity purification will also increase ([13], this volume). In the past the development of synthetic peptide substrates has lagged behind the discovery of protein kinases. This situation is changing due to the widespread use of many synthetic peptides, such as the kemptide and the ribosomal $6 peptide, which have been used to detect protein kinases with unforeseen overlapping specificities to the cAMP-dependent protein kinase. 75'76 The availability of relatively specific inhibitors such as the cyclic AMP-dependent protein kinase inhibitor peptide PKI(5-22) 77 and other pseudosubstrate inhibitors (this series, Volume, 201 [24]) as well as calcium chelators for calcium-dependent protein kinases greatly facilitates attempts to detect new protein kinase activities using peptide substrates capable of being phosphorylated by multiple protein kinases. It can be expected that in the forthcoming years there will be even greater synergy between the use of recombinant protein expression and synthetic peptides to create a variety of protein kinase substrates in order to explore the mechanisms of regulation by protein phosphorylation. 74 B. Luscher, E. Christenson, D. W. Litchfield, E. G. Krebs, and R. N. Eisenman, Nature (London) 344, 517 (1990). 75 E. Erikson and J. L. Mailer, Second Messengers Phosphoproteins 12, 135 (1988). 76 j. K. Klarlund, A. P. Bradford, M. G. Milla, and M. P. Czech, J. Biol. Chem. 265, 227 (1990). 77 B. E. Kemp, H. C. Cheng, and D. A. Walsh, this series, Vol. 159, p. 173.
[11] S y n t h e t i c P e p t i d e S u b s t r a t e s for C a s e i n K i n a s e II
By DANIEL R. MARSHAKand DENNIS CARROLL Casein Kinase II
The enzyme casein kinase II (CKII) is a protein-serine/threonine kinase found in all eukaryotic cells.~ Its ubiquitous distribution among species and tissues implies a function central to all nucleated cells. CKII was first identified from rabbit reticulocyte lysates, 2 and subsequently isolated from hypotonic extracts of mammalian liver and lung tissue) Following I G. M. Hathaway and J. A. Traugh, Curr. Top. Cell. Regul. 21, 101 (1982). 2 G. M. Hathaway and J. A. Traugh, J. Biol. Chem. 254, 762 (1979).
METHODS IN ENZYMOLOGY,VOL. 200
Copyright© 1991by AcademicPress, Inc. All rightsof reproductionin any formreserved.
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anion-exchange chromatography of these extracts on DEAE-cellulose and assay of the resulting fractions using partially hydrolyzed casein as substrate, two major peaks of casein kinase activity were identified. The species eluting at a higher salt concentration was designated CKII. Purification of CKII has been accomplished from various sources, including mammals, worms, flies, and yeast. The enzyme consists of two subunits, 0/and/3, with molecular weight ranges of 37K-44K and 24K-28K, respectively, with an apparent subunit composition of 0/2/32. The 0/subunit appears to be catalytic based on homology to other protein kinases, particularly in the consensus ATP-binding region, by affinity labeling the ATPbinding site, and by expression of the cDNA for the 0/subunit. 4 There appears to be a second form of the 0/ subunit, designated 0/', but the significance of this alternative structure is not yet clear. The role of the/3 subunit appears to be that of a regulatory element, as it is phosphorylated by exogenous kinases and contains sites of autophosphorylation. 5 The complete amino acid sequences of these subunits is available, either from protein structural determinations or inferred from cDNA structures. The enzyme is capable of using either ATP or GTP as substrate with similar K m values (10 and 30 /~M, respectively). The enzyme activity can be activated by polycationic materials, such as polyamines, and inhibited with low concentrations of polyanionic compounds, such as heparin. In recent years, CKII has been found to be stimulated by insulin 6or epidermal growth factor7 in cultured cells, by serum stimulation of quiescent fibroblasts, s by virus infection of epithelial c e l l s , 9 and by phorbol ester stimulation of primary cultures of kidney cells) ° These studies indicate that CKII is regulated in various cells, both by hormonal stimulation and by pharmacological agents. Many substrates have been identified for CKII, although the physiological relevance of the phosphorylation is not clear in all cases. Casein, the 3 A. M. Edelman, D. K. Blumenthal, and E. G. Krebs, Annu. Rev. Biochem. 56, 567 (1987). The characteristics of CKII referred to are summarized in this review. 4 R. Padmanabha, J. L. Chen-Wa, D. E. Hanna, and C. V. Glover, Mol. Cell. Biol. 10, 4089 (1990). 5 p. Ackerman, C. V. C. Glover, and N. Osheroff, Proc. Natl. Acad. Sci. U.S.A. 87, 821
(1990). 6 j. Sommercorn and E. G. Krebs, J. Biol. Chem. ,~2, 3839 (1987). 7 p. Ackerman and N. Osheroff, J. Biol. Chem. 264, 11958 (1989). s D. Carroll and D. R. Marshak, J. Biol. Chem. 264, 7345 (1989). 9 D. Carroll, E. Moran, and D. R. Marshak, unpublished observations (1988). l0 D. Carroll, N. Santoro, and D. R. Marshak, Cold Spring Harbor Syrup. Quant. Biol. 53, 91 (1988). Two points in this reference require clarification. The Vmx values quoted in this reference are in nmol/10 min/mg, and are therefore consistent with the results presented here. Also, the sequence of the Fos peptide was the 19-mer described here (Table I).
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ASSAYS OF PROTEIN KINASES
[1 1]
substrate first used for the enzyme, appears to be a nonphysiological substrate. The CKII enzyme activity is found both in cytosol and in nuclei, and there are substrates identified in both locations. Cytosolic substrates include proteins involved in translational control 1 (e.g., eukaryotic initiation factor (eIF)-2, -3, -4B, and -5), metabolic regulation (e.g., glycogen synthase II and calmodulin12), and the cytoskeleton (e.g. nonmuscle myosin, 13fl_tubulin14). Substrates found in the nucleus include DNA topoisomerase II, 15RNA polymerases I and II, 16oncoproteins I°A7As(e.g., adenovirus E l a, SV40 large T antigen, Myc, Fos, Myb), and transcription factors'9 (e.g., serum response factor, cAMP regulatory element-binding protein). The extraordinary range of substrates for this enzyme supports the contention that CKII plays a significant role in cell physiology. The specificity of CKII, like many other protein kinases, requires only a short recognition sequence that can be identified from the primary structure, including a serine or threonine, followed by a cluster of at least three acidic amino acids. 2°'21 Structures containing predicted/3 turns generally have a lower Km than those without such structures. 2°-22 For example, one or two proline residues preceding the phosphorylated serine or threonine are common in CKII substrates. Basic residues immediately adjacent to the site of phosphorylation reduce the performance of a substrate, but basic residues distal to the site may have some beneficial effect. This may relate to the activation properties of polycationic substances. In this chapter we review the use of new peptide substrates for CKII based on several of the nuclear oncoproteins. This class of CKII substrates is of special interest in transcriptional control, cell proliferation, tissue differentiation, and the pathophysiology of cancer. The methods presented permit the quantitative analysis of the phosphorylation of these model 11 C. Picton, J. Woodgett, B. Hemmings, and P. Cohen, FEBS Lett. 150, 191 (1982). 12 F. Meggio, A. M. Brunati, and L. A. Pinna, FEBS Left. 215, 241 (1987). t3 N. Murakami, G. Healy-Louie, and M. Elzinga, J. Biol. Chem. 265, 1041 (1990). t4 D. S. Kohtz and S. Puszkin, J. Neurochem. 52, 285 (1989). 15 p. Ackerman, C. V. C. GIover, and N. Osheroff, J. Biol. Chem. 263, 12653 (1988). 16 D. A. Stetler and K. M. Rose, Biochemistry 21, 3721 (1982). t7 B. Liischer, E. A. Kuenzel, E. G. Krebs, and R. N. Eisenman, E M B O J . 8, 1111 (1989). is B. Ltischer, E. Christenson, D. W. Litchfield, E. G. Krebs, and R. N. Eisenman, Nature (London) 344, 517 (1990). 19j. R. Manak, N. de Bisschop, R. M. Kris, and R. Prywes, Genes Deo. 4, 955 (1990). 20 F. Meggio, F. Marchiori, G. Borin, G. Chessa, and L. A. Pinna, J. Biol. Chem. 259, 14576 (1984). 21 E. A. Kuenzel, J. A. Mulligan, J. Sommercorn, and E. G. Krehs, J. Biol. Chem. 262, 9136 (1987). 22 D. Small, P. Y. Chou, and G. D. Fasman, Biochem. Biophys. Res. Commun. 79, 341 (1977).
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substrates. Throughout such studies, we advocate a multidisciplinary approach 23,24utilizing a variety of methodologies, such as peptide synthesis, high-performance liquid chromatography (HPLC), thin-layer chromatography, chemical modification, protein sequencing, amino acid analysis, and mass spectrometry. Through such an approach, complex questions can be addressed, such as the level of phosphorylation at adjacent sites, the order of addition of phosphates at multiple sites, and the identification of peptide contaminants in synthetic products. Synthetic Peptide Substrates Based on Oncoproteins Oncoproteins refer to the proteins products of oncogenes; that is, genes defined by mutations that affect the ability of those genes to transform cells in culture or to produce tumors in animals when introduced through viruses, artifically transfected cells, or external mutagenesis.25 Many of the known cytoplasmic oncoproteins affect some aspect of signal transduction mechanisms at the level of growth factors, receptors, G proteins, or protein kinases. The nuclear oncoproteins of viral and cellular origins are often DNA-binding proteins, transcription factors, and/or regulators of DNA replication. Through genetic analysis, nuclear oncoproteins have been found to have discrete domains that are responsible for cell transformation, cell proliferation, D N A binding, transcriptional control, or protein-protein interactions. 26'27 In several cases, these sites include sequences that meet the requirements for candidate CKII phosphorylation sites. Labeling studies using [3ap]phosphate indicate that most, if not all of these proteins are phosphorylated in vivo, and in some cases, peptide maps are consistent with the notion that CKII phosphorylates the expected site. 17'1s Several transcription factors that are either oncoproteins themselves or are involved in oncogene expression contain consensus sequences for CKII phosphorylation sites. These include the serum response factor (srf) and the cAMP response element-binding protein (creb) that regulate the f o s gene, zs the fos/jun protein families themselves, a9 and the yeast transcripD. R. Marshak and B. A. Fraser, in "Brain Peptides Update" (J. B. Martin, M. J. Brownstein, and D. T. Krieger, eds.), Vol. 1, p. 13. Wiley, New York, 1987. 24D. R. Marshak and B. A. Fraser, in "High Performance Liquid Chromatography in Biotechnology" (W. Hancock, ed.), pp. 531. Wiley, New York. 25j. M. Bishop, Annu. Rev. Biochem. 52, 301 (1983). 26j. Ma and M. Ptashne, Cell (Cambridge, Mass.) 48, 847 (1987). 27E. Moran and M. B. Matthews, Cell (Cambridge, Mass.) 48, 177 (1987). 28M. Gilman, R. N. Wilson, and R. A. Weinberg, Mol. Cell. Biol. 6, 4305 (1986). B. R. Franza, Jr., F. J. Ranscher, III, S. F. Josephs, and T. Curran, Science 239, 1150 (1988).
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ASSAYS OF PROTEIN KINASES
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TABLE I SYNTHETICPEPTIDE SUBSTRATESOF CASEINKINASEII Protein Ela LTag Myc Fos Nef ETE p53
Km
Vmax
(/xM)
(nmol/min/mg)
Sequence a
45 54 31 6 30 38 b
H-HEAGFPPSDDEDEEG-NH2 H-SEEMPSSDDEATAD-NH2 H-EEETPPTTSSDSEEEQEDEEE-oH H-RRGKVEQLSPEEEEKRRIRR-NH2 H-MDDVDSDDDD-NH2 H-RRREEETEEE-oH H-RRTEEE-o8
25 35 27 2 0.65 720 b
a A, Alanine;D, aspartic acid; E, glutamic acid; F, phenylalanine;G, glycine; H, histidine; I, isoleucine; L, leucine; M,methionine; P, protine; Q, glutamine; R, arginine; S, serine; T, threonine; V, valine. b The p53 peptide did not show significant incorporation of phosphate using bovine liver casein kinase II at peptide concentrations up to 10 raM.
tion factors GAL4 and GCN4. 26 In the case of srf, CKII phosphorylation appears to cause a more avid interaction with DNA,29 while Myb,lS Myc,30 and LTag 31display decreased binding to DNA after CKII phosphorylation. Whether these effects mimic the in vivo function of CKII is not yet established, but it is likely that each protein will have to be analyzed independently for the effects of CKII phosphorylation. We have used synthetic peptides as models of the phosphorylation sites for CKII, based on the primary sequences of adenovirus Ela, simian virus 40 large T antigen (LTag), the human immunodeficiency virus nef protein, the human antioncoprotein p53, Myc, Fos, and Myb. The sequence of these peptides are shown in Table I, along with the K mand Vm~, values for the peptides using purified bovine liver CKII. For comparison, the peptide substrate developed by Kuenzel and Krebs 32(RRREEETEEE) is shown, designated the ETE peptide. The kinetic parameters for the peptides vary over three orders of magnitude, but all appear to be specific substrates of CKII. and give values similar to those previously reported.2° Of note are the Fos and nefpeptides that have quite low K m values. The nef peptide is an outstanding substrate, displaying submicromolar K m values, and is similar to the DSD peptide used by Liischer et al) 8 (RRRDDDSDDD). The Fos peptide shows a low Vm~xvalue, as well as a low K m value, and displays inhil~ition of CKII phosphorylation at high 3oD. Carroll, W.-K. Chart, M. T. Vandenberg, D. Spector, and D. R. Marshak, in preparation. 31 D.Carroll, D. McVey, and D. R. Marshak, (1989) unpublished observations. 32E. A. Kuenzel and E. G. Krebs, Proc. Natl. Acad. Sci. U.S.A. 82, 737 (1985).
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SYNTHETICPEPTIDESUBSTRATESFOR CKII
139
concentrations. This observation led us to begin to examine peptides closely for the possibility of contamination with adducts or other modified synthetic materials. In addition, the reported Km value for the ETE model peptide varies slightly from that originally described,32 suggesting inconsistencies either in assay procedures or in the synthetic products from different laboratories. It is of interest to compare the ETE peptide with the p53 peptide, which is not a good substrate for CKII. The major differences in the two peptides is the overall size and the proximity of the arginine residues to the threonine residue. Therefore, we felt it necessary to eliminate the possibility that termination, deletion or other artifacts of the synthesis were not altering the apparent kinetic parameters for the peptide. Below we address problems in synthetic procedures for CKII substrate peptides, followed by a summary of assay protocols that have given reproducible results in our laboratory. S y n t h e s i s of P e p t i d e s
Chemical synthesis of peptides using automated instrumentation is convenient, rapid, and cost effective. However, the synthesis of peptide substrates for an enzyme, in this case CKII, requires attention to the details of the synthetic chemistry and significant purification and characterization of the product. It is essential to remove minor contaminants such as deletion products, termination products, incompletely deprotected analogs, scavenger adducts, and chemically modified forms. Such contaminants can lead to erroneous results and/or lot-to-lot variability when measuring the kinetics of peptide phosphorylation. For example, a modified peptide might act as a competitive inhibitor, and even a contaminant of 1% would seriously affect the kinetics if the Ki value is below the Km value of the major product. Therefore, crude peptides produced by automated synthesis without purification are not suitable for detailed kinetic analysis.
Automated Synthesis The synthetic chemistry employed for peptides in our laboratory utilizes an Applied Biosystems (Foster City, CA) 430A instrument to couple preformed, symmetric anhydrides or 1-hydroxybenzotriazole (HOB0-activated esters of N~-t-Boc amino acids on phenylacetamidomethyl-derivatized or benzhydrylamine-derivatizedpolystyrene resins (1% divinylbenzene cross-linked) at a substitution level >0.5 mmol/g. These procedures are based on the methods of Merrifield for solid-phase peptide synthesis)3 33G. Barany and R. B. Merrifield, in "The Peptides: Structure, Function, Biology" (E. Gross and J. Meienhofer, eds.), Vol. 2, p. 1. Academic Press, New York, 1980.
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ASSAYS OF PROTEIN KINASES
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Other chemistries, particularly that using 9-fluorenylmethyloxycarbonyl (Fmoc)-protected amino acids, are quite suitable for synthesis. Choice of solvent system is important to optimize yields of the desired peptide. Dichloromethane, dimethylformamide, N-methylpyrrolidone, and dimethyl sulfoxide are the most frequently employed. When using dichloromethane, it is useful to recouple each amino acid in a more polar solvent, such as dimethylformamide. Our work has been with N-methylpyrrolidone and dichloromethane, including an additional coupling period in dimethyl sulfoxide. Although reaction times are increased to about 90-120 min, these solvents promote swelling of the polystyrene, allowing a higher coupling yield. In addition, we routinely treat the resin with acetic anhydride following the completion of the coupling reaction to block any unreacted N~-amino groups and prevent further reaction. This technique resuits in the formation of a series of termination products with N ~acetylations rather than a large number of deletion products. Acetylated termination products are more easily separated by chromatography than are deletion products because termination peptides generally have shorter retention times compared to the desired product. Deprotection
Another area of concern is the modifications and side reactions that can occur during acidolytic cleavage of the peptide from the resin and deprotection of the side chains. Synthetic peptide substrates for CKII always include several aspartic and/or glutamic acid residues, and there are particular side reactions that plague these syntheses. Failure to compensate for these reactions will lead to peptide products that yield erroneous results on phosphorylation. Cleavage and deprotection of the peptide from the resin involve acidolytic treatment, usually in either anhydrous hydrogen fluoride or trifluoromethanesulfonic acid.34 The latter treatment is extremely dehydrating and can lead to the cyclization of acidic or amidated amino acids. In particular, the use of the O-benzyl-protecting group on aspartic acid can lead to the cyclization of the side chain to form the aspartimide. Using O-cycloalkanes as protecting groups for aspartic acid residues this side reaction because these esters are poor leaving groups compared to O-benzyl, and hydrolysis is favored over cyclization by the peptide backbone N" moiety. Therefore, we routinely use N ~ - t Boc-(fl-O-cyclohexyl)aspartic acid. The second most frequent problem with synthesis of acidic peptides is adduct formation between the peptide H. Yajima and N. Fujii, in "The Peptides: Analysis, Synthesis, Biology" (E. Gross and J. Meienhofer, eds.), Vol. 5, p. 66. Academic Press, New York, 1983.
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glutamate and glutamine side chains and scavenger molecules, such as anisole or cresol. These chemicals are necessary in acidolytic deprotection to guard against reattachment of protecting groups. However, anisole adducts with glutamyl residues can significantly alter the charge of the product and introduce an unwanted modification. Such adducts are not readily detectable on amino acid analysis because aqueous acid hydrolysis at elevated temperatures destroys the adduct, producing free glutamic acid. Thus, amino acid analysis aloneis not adequate documentation of the purity of a synthetic peptide.
Solubilization and Desalting Following deprotection, scavengers and other organic reaction products are removed by ether extraction and precipitation of the peptide. Solubilization of the precipitated peptide can be difficult, particularly for acidic CKII substrates. If the product is to be lyophilized, neutralizing the precipitate with a volatile base, such as ammonium hydroxide or triethylamine, is necessary. Variable proportions of an organic solvent, such as methanol or acetonitrile, are often required for solubilization of the neutralized peptide in aqueous media. Alternatively, the product can be neutralized with sodium hydroxide or Tris (trishydroxymethylaminomethane), followed by solubilization in aqueous 6 M guanidine-hydrochloride or 8 M urea. The choice of solubilization conditions depends on the desalting step to follow. Three options are available, all of which have advantages and disadvantages for CKII substrates. First, large-scale gel filtration can be used to remove small-molecule contaminants. We have used columns of 5.0 x 100 cm or greater in size containing polyacrylamide resin, BioGel P-2 (Bio-Rad, Richmond, CA). Solvents for these columns are either 0.05 M ammonium acetate or 0.1% acetic acid containing 20% (v/v) acetonitrile. Choice of solvents depends on the solubility of the particular peptide. When using gel filtration, it is usually necessary to concentrate the solubilized, ether-precipitated peptide through lyophilization. Therefore, a volatile solvent system must be used in solubilization. Second, large-scale, reversed-phase HPLC is very useful for desalting the peptide. We have used the Waters (Milford, MA) Delta Prep 3000 preparative HPLC equipped with the Prep-Pak module that permits cartridge columns of 4.8 × 30 cm. Flow rates are generally 25-75 ml/min with solvent consisting of 0.1% aqueous trifluoroacetic acid and increasing proportions of acetonitrile. The choice of C 4- or C is-modified silica depends on the size of the peptide; peptides
100 o
UJ Z
I
2
3
I
21100
I 2200
i
80
40
20 0 1500
1600
1700 '
1800 ' FIG.
19'0 0
2000
I 2300
'
(M/Z
3.(continued).
The serine at position 7, corresponding to residue 112, acquires phosphate with linear kinetics, while the serine at position 6, corresponding to residue 11 I, displays a lag, followed by a similar rate of phosphorylation. These results suggest the sequential addition of phosphate to these adjacent position. Once serine-112 is phosphorylated, the preceding serine becomes a substrate for CKII. There may be important implications of this ordered phosphorylation, particularly with respect to DNA replication and neighboring phosphorylation sites. A separate analysis of this domain on LTag revealed a downstream phosphorylation site at threonine-124 for the cell division cycle-regulated protein kinase, p34 cdc2. Phosphorylation at that site increases DNA replication by ~tering binding of LTag to the viral origin of replication. 42 The role of the neighboring CKII sites is not full understood. However, the use of synthetic peptide substrates, in conjunction with biochemical and genetic analysis of the intact protein, provides
[11]
SYNTHETIC PEP'rIDE SUBSTRATES FOR C K I I
155
S#EMPS6S' EATAO
50
I0~ . . . . . . . .
0
., . . . . . . . . .
2
Time of
s,
', . . . . . . . . .
4 Incubotion (hours)
6
FIG. 4. Ordered addition of phosphate to an LTag peptide (sequence shown in Table I). The peptide was phosphorylated with bovine liver CKII for various amounts of time as shown on the abscissa. Following the phosphorylation, the peptide was treated with 0.1 M Ba(OH) 2 and 1 M ethanethiol at 30* for 1 hr. Another aliquot of ethanethiol was added to bring the final concentration to 2 M, and the reaction was incubated for an additional 2 hr. The reaction was terminated by lowering the pH to 4.0 with acetic acid, and the peptide was desalted by reversed-phase HPLC. The modified peptide was then subjected to automated sequencing on an Applied Biosystems 470A instrument equipped with an on-line HPLC and data system. The Phenylthiohydantoin (Pth)-S-ethylcysteine at each cycle was quantitated by comparison to a standard synthesized in our laboratory.
a powerful approach to understanding nuclear oncoproteins.
the phosphorylation
domains of
Summary Syntheticpeptide substratesfor CKII are usefulreagentsin the analysis of phosphorylation siteswhen used in conjunction with biochemical and genetic analysisof the protein substratesfor the enzyme. A multidisciplinary approach should be applied to the characterizationof the synthetic
156
ASSAYS OF PROTEIN KINASES
[11]
peptide products, including amino acid analysis, sequencing, and mass spectrometry. Synthetic procedures for CKII substrate peptides often result in anisole adducts and dehydrated forms. Mass spectrometry is invaluable in identifying these contaminants, and preparative HPLC can be used to separate them from the desired product. Quantitative analysis of the CKII phosphorylation of peptides can utilize phosphocellulose paper if the peptide has a basic sequence, or thin-layer chromatography, if the peptide has no basic portion. Qualitative analysis using electrophoresis and mass spectrometry help to establish the stoichiometry ofphosphorylation. Sequence analysis of phosphoserine after/3 elimination and derivitization is useful in quantifying adjacent phosphorylation sites. Overall, application of a variety of techniques permits detailed analysis of CKII phosphorylation sites on synthetic peptides that are model substrates. Acknowledgments We are indebted to M. Vandenberg, G. Binns, M. MeneiUy,and N. Santoro for expert technical assistance. This work was supported by NIH Grants CA13106, CA45508, and CA09311 and NSF Grant BNS8707558.
ASSAYS OF PROTEIN KINASES
[1 1]
most likely s i t e . 74 The power of this approach can be further enhanced by making complementary point mutations in the protein substrate. With improvements in the potency of peptide substrates it is likely that applications of affinity purification will also increase ([13], this volume). In the past the development of synthetic peptide substrates has lagged behind the discovery of protein kinases. This situation is changing due to the widespread use of many synthetic peptides, such as the kemptide and the ribosomal $6 peptide, which have been used to detect protein kinases with unforeseen overlapping specificities to the cAMP-dependent protein kinase. 75'76 The availability of relatively specific inhibitors such as the cyclic AMP-dependent protein kinase inhibitor peptide PKI(5-22) 77 and other pseudosubstrate inhibitors (this series, Volume, 201 [24]) as well as calcium chelators for calcium-dependent protein kinases greatly facilitates attempts to detect new protein kinase activities using peptide substrates capable of being phosphorylated by multiple protein kinases. It can be expected that in the forthcoming years there will be even greater synergy between the use of recombinant protein expression and synthetic peptides to create a variety of protein kinase substrates in order to explore the mechanisms of regulation by protein phosphorylation. 74 B. Luscher, E. Christenson, D. W. Litchfield, E. G. Krebs, and R. N. Eisenman, Nature (London) 344, 517 (1990). 75 E. Erikson and J. L. Mailer, Second Messengers Phosphoproteins 12, 135 (1988). 76 j. K. Klarlund, A. P. Bradford, M. G. Milla, and M. P. Czech, J. Biol. Chem. 265, 227 (1990). 77 B. E. Kemp, H. C. Cheng, and D. A. Walsh, this series, Vol. 159, p. 173.
[11] S y n t h e t i c P e p t i d e S u b s t r a t e s for C a s e i n K i n a s e II
By DANIEL R. MARSHAKand DENNIS CARROLL Casein Kinase II
The enzyme casein kinase II (CKII) is a protein-serine/threonine kinase found in all eukaryotic cells.~ Its ubiquitous distribution among species and tissues implies a function central to all nucleated cells. CKII was first identified from rabbit reticulocyte lysates, 2 and subsequently isolated from hypotonic extracts of mammalian liver and lung tissue) Following I G. M. Hathaway and J. A. Traugh, Curr. Top. Cell. Regul. 21, 101 (1982). 2 G. M. Hathaway and J. A. Traugh, J. Biol. Chem. 254, 762 (1979).
METHODS IN ENZYMOLOGY,VOL. 200
Copyright© 1991by AcademicPress, Inc. All rightsof reproductionin any formreserved.
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135
anion-exchange chromatography of these extracts on DEAE-cellulose and assay of the resulting fractions using partially hydrolyzed casein as substrate, two major peaks of casein kinase activity were identified. The species eluting at a higher salt concentration was designated CKII. Purification of CKII has been accomplished from various sources, including mammals, worms, flies, and yeast. The enzyme consists of two subunits, 0/and/3, with molecular weight ranges of 37K-44K and 24K-28K, respectively, with an apparent subunit composition of 0/2/32. The 0/subunit appears to be catalytic based on homology to other protein kinases, particularly in the consensus ATP-binding region, by affinity labeling the ATPbinding site, and by expression of the cDNA for the 0/subunit. 4 There appears to be a second form of the 0/ subunit, designated 0/', but the significance of this alternative structure is not yet clear. The role of the/3 subunit appears to be that of a regulatory element, as it is phosphorylated by exogenous kinases and contains sites of autophosphorylation. 5 The complete amino acid sequences of these subunits is available, either from protein structural determinations or inferred from cDNA structures. The enzyme is capable of using either ATP or GTP as substrate with similar K m values (10 and 30 /~M, respectively). The enzyme activity can be activated by polycationic materials, such as polyamines, and inhibited with low concentrations of polyanionic compounds, such as heparin. In recent years, CKII has been found to be stimulated by insulin 6or epidermal growth factor7 in cultured cells, by serum stimulation of quiescent fibroblasts, s by virus infection of epithelial c e l l s , 9 and by phorbol ester stimulation of primary cultures of kidney cells) ° These studies indicate that CKII is regulated in various cells, both by hormonal stimulation and by pharmacological agents. Many substrates have been identified for CKII, although the physiological relevance of the phosphorylation is not clear in all cases. Casein, the 3 A. M. Edelman, D. K. Blumenthal, and E. G. Krebs, Annu. Rev. Biochem. 56, 567 (1987). The characteristics of CKII referred to are summarized in this review. 4 R. Padmanabha, J. L. Chen-Wa, D. E. Hanna, and C. V. Glover, Mol. Cell. Biol. 10, 4089 (1990). 5 p. Ackerman, C. V. C. Glover, and N. Osheroff, Proc. Natl. Acad. Sci. U.S.A. 87, 821
(1990). 6 j. Sommercorn and E. G. Krebs, J. Biol. Chem. ,~2, 3839 (1987). 7 p. Ackerman and N. Osheroff, J. Biol. Chem. 264, 11958 (1989). s D. Carroll and D. R. Marshak, J. Biol. Chem. 264, 7345 (1989). 9 D. Carroll, E. Moran, and D. R. Marshak, unpublished observations (1988). l0 D. Carroll, N. Santoro, and D. R. Marshak, Cold Spring Harbor Syrup. Quant. Biol. 53, 91 (1988). Two points in this reference require clarification. The Vmx values quoted in this reference are in nmol/10 min/mg, and are therefore consistent with the results presented here. Also, the sequence of the Fos peptide was the 19-mer described here (Table I).
136
ASSAYS OF PROTEIN KINASES
[1 1]
substrate first used for the enzyme, appears to be a nonphysiological substrate. The CKII enzyme activity is found both in cytosol and in nuclei, and there are substrates identified in both locations. Cytosolic substrates include proteins involved in translational control 1 (e.g., eukaryotic initiation factor (eIF)-2, -3, -4B, and -5), metabolic regulation (e.g., glycogen synthase II and calmodulin12), and the cytoskeleton (e.g. nonmuscle myosin, 13fl_tubulin14). Substrates found in the nucleus include DNA topoisomerase II, 15RNA polymerases I and II, 16oncoproteins I°A7As(e.g., adenovirus E l a, SV40 large T antigen, Myc, Fos, Myb), and transcription factors'9 (e.g., serum response factor, cAMP regulatory element-binding protein). The extraordinary range of substrates for this enzyme supports the contention that CKII plays a significant role in cell physiology. The specificity of CKII, like many other protein kinases, requires only a short recognition sequence that can be identified from the primary structure, including a serine or threonine, followed by a cluster of at least three acidic amino acids. 2°'21 Structures containing predicted/3 turns generally have a lower Km than those without such structures. 2°-22 For example, one or two proline residues preceding the phosphorylated serine or threonine are common in CKII substrates. Basic residues immediately adjacent to the site of phosphorylation reduce the performance of a substrate, but basic residues distal to the site may have some beneficial effect. This may relate to the activation properties of polycationic substances. In this chapter we review the use of new peptide substrates for CKII based on several of the nuclear oncoproteins. This class of CKII substrates is of special interest in transcriptional control, cell proliferation, tissue differentiation, and the pathophysiology of cancer. The methods presented permit the quantitative analysis of the phosphorylation of these model 11 C. Picton, J. Woodgett, B. Hemmings, and P. Cohen, FEBS Lett. 150, 191 (1982). 12 F. Meggio, A. M. Brunati, and L. A. Pinna, FEBS Left. 215, 241 (1987). t3 N. Murakami, G. Healy-Louie, and M. Elzinga, J. Biol. Chem. 265, 1041 (1990). t4 D. S. Kohtz and S. Puszkin, J. Neurochem. 52, 285 (1989). 15 p. Ackerman, C. V. C. GIover, and N. Osheroff, J. Biol. Chem. 263, 12653 (1988). 16 D. A. Stetler and K. M. Rose, Biochemistry 21, 3721 (1982). t7 B. Liischer, E. A. Kuenzel, E. G. Krebs, and R. N. Eisenman, E M B O J . 8, 1111 (1989). is B. Ltischer, E. Christenson, D. W. Litchfield, E. G. Krebs, and R. N. Eisenman, Nature (London) 344, 517 (1990). 19j. R. Manak, N. de Bisschop, R. M. Kris, and R. Prywes, Genes Deo. 4, 955 (1990). 20 F. Meggio, F. Marchiori, G. Borin, G. Chessa, and L. A. Pinna, J. Biol. Chem. 259, 14576 (1984). 21 E. A. Kuenzel, J. A. Mulligan, J. Sommercorn, and E. G. Krehs, J. Biol. Chem. 262, 9136 (1987). 22 D. Small, P. Y. Chou, and G. D. Fasman, Biochem. Biophys. Res. Commun. 79, 341 (1977).
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137
substrates. Throughout such studies, we advocate a multidisciplinary approach 23,24utilizing a variety of methodologies, such as peptide synthesis, high-performance liquid chromatography (HPLC), thin-layer chromatography, chemical modification, protein sequencing, amino acid analysis, and mass spectrometry. Through such an approach, complex questions can be addressed, such as the level of phosphorylation at adjacent sites, the order of addition of phosphates at multiple sites, and the identification of peptide contaminants in synthetic products. Synthetic Peptide Substrates Based on Oncoproteins Oncoproteins refer to the proteins products of oncogenes; that is, genes defined by mutations that affect the ability of those genes to transform cells in culture or to produce tumors in animals when introduced through viruses, artifically transfected cells, or external mutagenesis.25 Many of the known cytoplasmic oncoproteins affect some aspect of signal transduction mechanisms at the level of growth factors, receptors, G proteins, or protein kinases. The nuclear oncoproteins of viral and cellular origins are often DNA-binding proteins, transcription factors, and/or regulators of DNA replication. Through genetic analysis, nuclear oncoproteins have been found to have discrete domains that are responsible for cell transformation, cell proliferation, D N A binding, transcriptional control, or protein-protein interactions. 26'27 In several cases, these sites include sequences that meet the requirements for candidate CKII phosphorylation sites. Labeling studies using [3ap]phosphate indicate that most, if not all of these proteins are phosphorylated in vivo, and in some cases, peptide maps are consistent with the notion that CKII phosphorylates the expected site. 17'1s Several transcription factors that are either oncoproteins themselves or are involved in oncogene expression contain consensus sequences for CKII phosphorylation sites. These include the serum response factor (srf) and the cAMP response element-binding protein (creb) that regulate the f o s gene, zs the fos/jun protein families themselves, a9 and the yeast transcripD. R. Marshak and B. A. Fraser, in "Brain Peptides Update" (J. B. Martin, M. J. Brownstein, and D. T. Krieger, eds.), Vol. 1, p. 13. Wiley, New York, 1987. 24D. R. Marshak and B. A. Fraser, in "High Performance Liquid Chromatography in Biotechnology" (W. Hancock, ed.), pp. 531. Wiley, New York. 25j. M. Bishop, Annu. Rev. Biochem. 52, 301 (1983). 26j. Ma and M. Ptashne, Cell (Cambridge, Mass.) 48, 847 (1987). 27E. Moran and M. B. Matthews, Cell (Cambridge, Mass.) 48, 177 (1987). 28M. Gilman, R. N. Wilson, and R. A. Weinberg, Mol. Cell. Biol. 6, 4305 (1986). B. R. Franza, Jr., F. J. Ranscher, III, S. F. Josephs, and T. Curran, Science 239, 1150 (1988).
138
ASSAYS OF PROTEIN KINASES
[1 1]
TABLE I SYNTHETICPEPTIDE SUBSTRATESOF CASEINKINASEII Protein Ela LTag Myc Fos Nef ETE p53
Km
Vmax
(/xM)
(nmol/min/mg)
Sequence a
45 54 31 6 30 38 b
H-HEAGFPPSDDEDEEG-NH2 H-SEEMPSSDDEATAD-NH2 H-EEETPPTTSSDSEEEQEDEEE-oH H-RRGKVEQLSPEEEEKRRIRR-NH2 H-MDDVDSDDDD-NH2 H-RRREEETEEE-oH H-RRTEEE-o8
25 35 27 2 0.65 720 b
a A, Alanine;D, aspartic acid; E, glutamic acid; F, phenylalanine;G, glycine; H, histidine; I, isoleucine; L, leucine; M,methionine; P, protine; Q, glutamine; R, arginine; S, serine; T, threonine; V, valine. b The p53 peptide did not show significant incorporation of phosphate using bovine liver casein kinase II at peptide concentrations up to 10 raM.
tion factors GAL4 and GCN4. 26 In the case of srf, CKII phosphorylation appears to cause a more avid interaction with DNA,29 while Myb,lS Myc,30 and LTag 31display decreased binding to DNA after CKII phosphorylation. Whether these effects mimic the in vivo function of CKII is not yet established, but it is likely that each protein will have to be analyzed independently for the effects of CKII phosphorylation. We have used synthetic peptides as models of the phosphorylation sites for CKII, based on the primary sequences of adenovirus Ela, simian virus 40 large T antigen (LTag), the human immunodeficiency virus nef protein, the human antioncoprotein p53, Myc, Fos, and Myb. The sequence of these peptides are shown in Table I, along with the K mand Vm~, values for the peptides using purified bovine liver CKII. For comparison, the peptide substrate developed by Kuenzel and Krebs 32(RRREEETEEE) is shown, designated the ETE peptide. The kinetic parameters for the peptides vary over three orders of magnitude, but all appear to be specific substrates of CKII. and give values similar to those previously reported.2° Of note are the Fos and nefpeptides that have quite low K m values. The nef peptide is an outstanding substrate, displaying submicromolar K m values, and is similar to the DSD peptide used by Liischer et al) 8 (RRRDDDSDDD). The Fos peptide shows a low Vm~xvalue, as well as a low K m value, and displays inhil~ition of CKII phosphorylation at high 3oD. Carroll, W.-K. Chart, M. T. Vandenberg, D. Spector, and D. R. Marshak, in preparation. 31 D.Carroll, D. McVey, and D. R. Marshak, (1989) unpublished observations. 32E. A. Kuenzel and E. G. Krebs, Proc. Natl. Acad. Sci. U.S.A. 82, 737 (1985).
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concentrations. This observation led us to begin to examine peptides closely for the possibility of contamination with adducts or other modified synthetic materials. In addition, the reported Km value for the ETE model peptide varies slightly from that originally described,32 suggesting inconsistencies either in assay procedures or in the synthetic products from different laboratories. It is of interest to compare the ETE peptide with the p53 peptide, which is not a good substrate for CKII. The major differences in the two peptides is the overall size and the proximity of the arginine residues to the threonine residue. Therefore, we felt it necessary to eliminate the possibility that termination, deletion or other artifacts of the synthesis were not altering the apparent kinetic parameters for the peptide. Below we address problems in synthetic procedures for CKII substrate peptides, followed by a summary of assay protocols that have given reproducible results in our laboratory. S y n t h e s i s of P e p t i d e s
Chemical synthesis of peptides using automated instrumentation is convenient, rapid, and cost effective. However, the synthesis of peptide substrates for an enzyme, in this case CKII, requires attention to the details of the synthetic chemistry and significant purification and characterization of the product. It is essential to remove minor contaminants such as deletion products, termination products, incompletely deprotected analogs, scavenger adducts, and chemically modified forms. Such contaminants can lead to erroneous results and/or lot-to-lot variability when measuring the kinetics of peptide phosphorylation. For example, a modified peptide might act as a competitive inhibitor, and even a contaminant of 1% would seriously affect the kinetics if the Ki value is below the Km value of the major product. Therefore, crude peptides produced by automated synthesis without purification are not suitable for detailed kinetic analysis.
Automated Synthesis The synthetic chemistry employed for peptides in our laboratory utilizes an Applied Biosystems (Foster City, CA) 430A instrument to couple preformed, symmetric anhydrides or 1-hydroxybenzotriazole (HOB0-activated esters of N~-t-Boc amino acids on phenylacetamidomethyl-derivatized or benzhydrylamine-derivatizedpolystyrene resins (1% divinylbenzene cross-linked) at a substitution level >0.5 mmol/g. These procedures are based on the methods of Merrifield for solid-phase peptide synthesis)3 33G. Barany and R. B. Merrifield, in "The Peptides: Structure, Function, Biology" (E. Gross and J. Meienhofer, eds.), Vol. 2, p. 1. Academic Press, New York, 1980.
140
ASSAYS OF PROTEIN KINASES
[11]
Other chemistries, particularly that using 9-fluorenylmethyloxycarbonyl (Fmoc)-protected amino acids, are quite suitable for synthesis. Choice of solvent system is important to optimize yields of the desired peptide. Dichloromethane, dimethylformamide, N-methylpyrrolidone, and dimethyl sulfoxide are the most frequently employed. When using dichloromethane, it is useful to recouple each amino acid in a more polar solvent, such as dimethylformamide. Our work has been with N-methylpyrrolidone and dichloromethane, including an additional coupling period in dimethyl sulfoxide. Although reaction times are increased to about 90-120 min, these solvents promote swelling of the polystyrene, allowing a higher coupling yield. In addition, we routinely treat the resin with acetic anhydride following the completion of the coupling reaction to block any unreacted N~-amino groups and prevent further reaction. This technique resuits in the formation of a series of termination products with N ~acetylations rather than a large number of deletion products. Acetylated termination products are more easily separated by chromatography than are deletion products because termination peptides generally have shorter retention times compared to the desired product. Deprotection
Another area of concern is the modifications and side reactions that can occur during acidolytic cleavage of the peptide from the resin and deprotection of the side chains. Synthetic peptide substrates for CKII always include several aspartic and/or glutamic acid residues, and there are particular side reactions that plague these syntheses. Failure to compensate for these reactions will lead to peptide products that yield erroneous results on phosphorylation. Cleavage and deprotection of the peptide from the resin involve acidolytic treatment, usually in either anhydrous hydrogen fluoride or trifluoromethanesulfonic acid.34 The latter treatment is extremely dehydrating and can lead to the cyclization of acidic or amidated amino acids. In particular, the use of the O-benzyl-protecting group on aspartic acid can lead to the cyclization of the side chain to form the aspartimide. Using O-cycloalkanes as protecting groups for aspartic acid residues this side reaction because these esters are poor leaving groups compared to O-benzyl, and hydrolysis is favored over cyclization by the peptide backbone N" moiety. Therefore, we routinely use N ~ - t Boc-(fl-O-cyclohexyl)aspartic acid. The second most frequent problem with synthesis of acidic peptides is adduct formation between the peptide H. Yajima and N. Fujii, in "The Peptides: Analysis, Synthesis, Biology" (E. Gross and J. Meienhofer, eds.), Vol. 5, p. 66. Academic Press, New York, 1983.
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SYNTHETICPEPTIDESUBSTRATESFOR CKII
141
glutamate and glutamine side chains and scavenger molecules, such as anisole or cresol. These chemicals are necessary in acidolytic deprotection to guard against reattachment of protecting groups. However, anisole adducts with glutamyl residues can significantly alter the charge of the product and introduce an unwanted modification. Such adducts are not readily detectable on amino acid analysis because aqueous acid hydrolysis at elevated temperatures destroys the adduct, producing free glutamic acid. Thus, amino acid analysis aloneis not adequate documentation of the purity of a synthetic peptide.
Solubilization and Desalting Following deprotection, scavengers and other organic reaction products are removed by ether extraction and precipitation of the peptide. Solubilization of the precipitated peptide can be difficult, particularly for acidic CKII substrates. If the product is to be lyophilized, neutralizing the precipitate with a volatile base, such as ammonium hydroxide or triethylamine, is necessary. Variable proportions of an organic solvent, such as methanol or acetonitrile, are often required for solubilization of the neutralized peptide in aqueous media. Alternatively, the product can be neutralized with sodium hydroxide or Tris (trishydroxymethylaminomethane), followed by solubilization in aqueous 6 M guanidine-hydrochloride or 8 M urea. The choice of solubilization conditions depends on the desalting step to follow. Three options are available, all of which have advantages and disadvantages for CKII substrates. First, large-scale gel filtration can be used to remove small-molecule contaminants. We have used columns of 5.0 x 100 cm or greater in size containing polyacrylamide resin, BioGel P-2 (Bio-Rad, Richmond, CA). Solvents for these columns are either 0.05 M ammonium acetate or 0.1% acetic acid containing 20% (v/v) acetonitrile. Choice of solvents depends on the solubility of the particular peptide. When using gel filtration, it is usually necessary to concentrate the solubilized, ether-precipitated peptide through lyophilization. Therefore, a volatile solvent system must be used in solubilization. Second, large-scale, reversed-phase HPLC is very useful for desalting the peptide. We have used the Waters (Milford, MA) Delta Prep 3000 preparative HPLC equipped with the Prep-Pak module that permits cartridge columns of 4.8 × 30 cm. Flow rates are generally 25-75 ml/min with solvent consisting of 0.1% aqueous trifluoroacetic acid and increasing proportions of acetonitrile. The choice of C 4- or C is-modified silica depends on the size of the peptide; peptides
100 o
UJ Z
I
2
3
I
21100
I 2200
i
80
40
20 0 1500
1600
1700 '
1800 ' FIG.
19'0 0
2000
I 2300
'
(M/Z
3.(continued).
The serine at position 7, corresponding to residue 112, acquires phosphate with linear kinetics, while the serine at position 6, corresponding to residue 11 I, displays a lag, followed by a similar rate of phosphorylation. These results suggest the sequential addition of phosphate to these adjacent position. Once serine-112 is phosphorylated, the preceding serine becomes a substrate for CKII. There may be important implications of this ordered phosphorylation, particularly with respect to DNA replication and neighboring phosphorylation sites. A separate analysis of this domain on LTag revealed a downstream phosphorylation site at threonine-124 for the cell division cycle-regulated protein kinase, p34 cdc2. Phosphorylation at that site increases DNA replication by ~tering binding of LTag to the viral origin of replication. 42 The role of the neighboring CKII sites is not full understood. However, the use of synthetic peptide substrates, in conjunction with biochemical and genetic analysis of the intact protein, provides
[11]
SYNTHETIC PEP'rIDE SUBSTRATES FOR C K I I
155
S#EMPS6S' EATAO
50
I0~ . . . . . . . .
0
., . . . . . . . . .
2
Time of
s,
', . . . . . . . . .
4 Incubotion (hours)
6
FIG. 4. Ordered addition of phosphate to an LTag peptide (sequence shown in Table I). The peptide was phosphorylated with bovine liver CKII for various amounts of time as shown on the abscissa. Following the phosphorylation, the peptide was treated with 0.1 M Ba(OH) 2 and 1 M ethanethiol at 30* for 1 hr. Another aliquot of ethanethiol was added to bring the final concentration to 2 M, and the reaction was incubated for an additional 2 hr. The reaction was terminated by lowering the pH to 4.0 with acetic acid, and the peptide was desalted by reversed-phase HPLC. The modified peptide was then subjected to automated sequencing on an Applied Biosystems 470A instrument equipped with an on-line HPLC and data system. The Phenylthiohydantoin (Pth)-S-ethylcysteine at each cycle was quantitated by comparison to a standard synthesized in our laboratory.
a powerful approach to understanding nuclear oncoproteins.
the phosphorylation
domains of
Summary Syntheticpeptide substratesfor CKII are usefulreagentsin the analysis of phosphorylation siteswhen used in conjunction with biochemical and genetic analysisof the protein substratesfor the enzyme. A multidisciplinary approach should be applied to the characterizationof the synthetic
156
ASSAYS OF PROTEIN KINASES
[11]
peptide products, including amino acid analysis, sequencing, and mass spectrometry. Synthetic procedures for CKII substrate peptides often result in anisole adducts and dehydrated forms. Mass spectrometry is invaluable in identifying these contaminants, and preparative HPLC can be used to separate them from the desired product. Quantitative analysis of the CKII phosphorylation of peptides can utilize phosphocellulose paper if the peptide has a basic sequence, or thin-layer chromatography, if the peptide has no basic portion. Qualitative analysis using electrophoresis and mass spectrometry help to establish the stoichiometry ofphosphorylation. Sequence analysis of phosphoserine after/3 elimination and derivitization is useful in quantifying adjacent phosphorylation sites. Overall, application of a variety of techniques permits detailed analysis of CKII phosphorylation sites on synthetic peptides that are model substrates. Acknowledgments We are indebted to M. Vandenberg, G. Binns, M. MeneiUy,and N. Santoro for expert technical assistance. This work was supported by NIH Grants CA13106, CA45508, and CA09311 and NSF Grant BNS8707558.
