Site-specific phosphorylation of the purified receptor for calcium-channel blockers by cAMP- and cGMP-dependent protein kinases, protein kinase C, calmodulin-dependent protein kinase II and casein kinase II

Five protein kinases were used to study the phosphorylation pattern of the purified skeletal muscle receptor for calcium-channel blockers (CaCB). cAMP kinase, cGMP kinase, protein kinase C, calmodulin kinase II and casein kinase II phosphorylated the 165-kDa and the 55-kDa proteins of the purified CaCB receptor. The 130/28-kDa and the 32-kDa protein of the receptor are not phosphorylated by these protein kinases. Among these protein kinases only cAMP kinase phosphorylated the 165-kDa subunit with 2-3-fold higher initial rate than the 55-kDa subunit. Casein kinase II phosphorylated the 165-kDa and the 55-kDa protein of the receptor with comparable rates. cGMP kinase, protein kinase C and calmodulin kinase II phosphorylated preferentially the 55-kDa protein. The 55-kDa protein is phosphorylated 50 times faster by cGMP kinase and protein kinase C than by calmodulin kinase II or casein kinase II and about 10 times faster by these enzymes than by cAMP kinase. Two-dimensional peptide maps of the 165-kDa subunit yielded a total of 11 phosphopeptides. Four or five peptides are phosphorylated specifically by cAMP kinase, cGMP kinase, casein kinase II and protein kinase C, whereas the other peptides are modified by several kinases. The same kinases phosphorylate 11 peptides in the 55-kDa subunit. Again, some of these peptides are modified specifically by each kinase. These results suggest that the 165-kDa and the 55-kDa subunit contain specific phosphorylation sites for cAMP kinase, cGMP kinase, casein kinase II and protein kinase C. Phosphorylation of these sites may be relevant for the in vivo function of the CaCB receptor.     

Phosphorylation of DARPP-32, a dopamine- and cAMP-regulated phosphoprotein, by casein kinase II

DARPP-32 (dopamine- and cAMP-regulated phosphorprotein, Mr = 32,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) is an inhibitor of protein phosphatase-1 and is enriched in dopaminoceptive neurons possessing the D1 dopamine receptor. Purified bovine DARPP-32 was phosphorylated in vitro by casein kinase II to a stoichiometry greater than 2 mol of phosphate/mol of protein whereas two structurally and functionally related proteins, protein phosphatase inhibitor-1 and G-substrate, were poor substrates for this enzyme. Sequencing of chymotryptic and thermolytic phosphopeptides from bovine DARPP-32 phosphorylated by casein kinase II suggested that the main phosphorylated residues were Ser45 and Ser102. In the case of rat DARPP-32, the identification of these phosphorylation sites was confirmed by manual Edman degradation. The phosphorylated residues are located NH2-terminal to acidic amino acid residues, a characteristic of casein kinase II phosphorylation sites. Casein kinase II phosphorylated DARPP-32 with an apparent Km value of 3.4 microM and a kcat value of 0.32 s-1. The kcat value for phosphorylation of Ser102 was 5-6 times greater than that for Ser45. Studies employing synthetic peptides encompassing each phosphorylation site confirmed this difference between the kcat values for phosphorylation of the two sites. In slices of rat caudate-putamen prelabeled with [32P]phosphate, DARPP-32 was phosphorylated on seryl residues under basal conditions. Comparison of thermolytic phosphopeptide maps and determination of the phosphorylated residue by manual Edman degradation identified the main phosphorylation site in intact cells as Ser102. In vitro, DARPP-32 phosphorylated by casein kinase II was dephosphorylated by protein phosphatases-1 and -2A. Phosphorylation by casein kinase II did not affect the potency of DARPP-32 as an inhibitor of protein phosphatase-1, which depended only on phosphorylation of Thr34 by cAMP-dependent protein kinase. However, phosphorylation of DARPP-32 by casein kinase II facilitated phosphorylation of Thr34 by cAMP-dependent protein kinase with a 2.2-fold increase in the Vmax and a 1.4-fold increase in the apparent Km. Phosphorylation of DARPP-32 by casein kinase II in intact cells may therefore modulate its phosphorylation in response to increased levels of cAMP.     

M-phase-specific cdc2 protein kinase phosphorylates the beta subunit of casein kinase II and increases casein kinase II activity

The M-phase-specific cdc2 (cell division control) protein kinase (a component of the M-phase-promoting factor) was found to activate casein kinase II in vitro. The increase in casein kinase II activity ranged over 1.5-5-fold. Increase in activity was prevented if ATP was replaced during the activation reaction by a non-hydrolysable analogue. Alkaline phosphatase treatment of the activated enzyme decreased the activity to the basal level. The beta subunit of casein kinase II was phosphorylated by cdc2 protein kinase at site(s) different from the autophosphorylation sites of the enzyme. Phosphoamino acid analysis showed that the beta subunit was phosphorylated by cdc2 protein kinase at threonine residues while autophosphorylation involved serine residues. Casein kinase II may be part of the cascade which leads to increased phosphorylation of many proteins at M-phase and therefore be involved in the pleiotropic effects of M-phase-promoting factor.     

Phosphorylation of varicella-zoster virus glycoprotein gpI by mammalian casein kinase II and casein kinase I.

Varicella-zoster virus (VZV) glycoprotein gpI is the predominant viral glycoprotein within the plasma membranes of infected cells. This viral glycoprotein is phosphorylated on its polypeptide backbone during biosynthesis. In this report, we investigated the protein kinases which participate in the phosphorylation events. Under in vivo conditions, VZV gpI was phosphorylated on its serine and threonine residues by protein kinases present within lysates of either VZV-infected or uninfected cells. Because this activity was diminished by heparin, a known inhibitor of casein kinase II, isolated gpI was incubated with purified casein kinase II and shown to be phosphorylated in an in vitro assay containing [gamma-32P]ATP. The same glycoprotein was phosphorylated when [32P]GTP was substituted for [32P]ATP in the protein kinase assay. We also tested whether VZV gpI was phosphorylated by two other ubiquitous mammalian protein kinases--casein kinase I and cyclic AMP-dependent kinase--and found that only casein kinase I modified gpI. When the predicted 623-amino-acid sequence of gpI was examined, two phosphorylation sites known to be optimal for casein kinase II were observed. Immediately upstream from each of the casein kinase II sites was a potential casein kinase I phosphorylation site. In summary, this study showed that VZV gpI was phosphorylated by each of two mammalian protein kinases (casein kinase I and casein kinase II) and that potential serine-threonine phosphorylation sites for each of these two kinases were present in the viral glycoprotein.     

Phosphorylation of heterochromatin protein 1 by casein kinase II is required for efficient heterochromatin binding in Drosophila.

Heterochromatin-associated protein 1 (HP1) is a nonhistone chromosomal protein with a dose-dependent effect on heterochromatin mediated position-effect silencing. It is multiply phosphorylated in vivo. Hyperphosphorylation of HP1 is correlated with heterochromatin assembly. We report here that HP1 is phosphorylated by casein kinase II in vivo at three serine residues located at the N and C termini of the protein. Alanine substitution mutations in the casein kinase II target phosphorylation sites dramatically reduce the heterochromatin binding activity of HP1, whereas glutamate substitution mutations, which mimic the charge contributions of phosphorylated serine, have apparently wild-type binding activity. We propose that phosphorylation of HP1 promotes protein-protein interaction between HP1 and target binding proteins in heterochromatin.     

Protein phosphatases active on acetyl-CoA carboxylase phosphorylated by casein kinase I, casein kinase II and the cAMP-dependent protein kinase

The protein phosphatases in rat liver cytosol, active on rat liver acetyl-CoA carboxylase (ACC) phosphorylated by casein kinase I, casein kinase II and the cAMP-dependent protein kinase, have been partially purified by anion-exchange and gel filtration chromatography. The major phosphatase activities against all three substrates copurify through fractionation and appear to be identical to protein phosphatases 2A1 and 2A2. No unique protein phosphatase active on 32P-ACC phosphorylated by the casein kinases was identified.     

Phosphorylation of nucleolin by a nucleolar type NII protein kinase

Nucleolin [C23 or 100 kilodaltons (kDa)] is the major nucleolar phosphorylated protein in exponentially growing Chinese hamster ovary cells. A nucleolar cyclic nucleotide independent protein kinase copurified with nucleolin in a complex which could be dissociated by hydroxyapatite chromatography. The kinase was stimulated by spermine and inhibited by heparin and presented most of the properties of nuclear casein kinase NII. Kinetic analyses showed the apparent Km value for nucleolin (7 X 10(-4) mg/mL) to be lower than those for other casein kinase II substrates such as nuclear protein HMG 14 (0.15 mg/mL), topoisomerase I (0.025 mg/mL), or topoisomerase II (0.04 mg/mL). Similarly, Vmax values were higher for nucleolin than for other substrates. Nucleolin thus appears to be a natural preferential substrate of nucleolar casein kinase NII. The kinase phosphorylated nucleolin in vitro at serine residues in a 29-kDa CNBr fragment located near the amino terminus of the molecule. The enzyme labeled typical casein kinase II sites. These sites were found predominantly in two highly acidic tryptic fragments designated A (residues 21-49) and C (residues 180-221) which contained serines having at least two acidic residues on their carboxyl-terminal sides. These results demonstrate the existence in the nucleolus of a type of NII protein kinase that uses a protein involved in ribosome assembly as preferential substrate.     

Phosphorylation of connexin 32, a hepatocyte gap-junction protein, by cAMP-dependent protein kinase, protein kinase C and Ca2+/calmodulin-dependent protein kinase II

Phosphorylation of connexin 32, the major liver gap-junction protein, was studied in purified liver gap junctions and in hepatocytes. In isolated gap junctions, connexin 32 was phosphorylated by cAMP-dependent protein kinase (cAMP-PK), by protein kinase C (PKC) and by Ca2+/calmodulin-dependent protein kinase II (Ca2+/CaM-PK II). Connexin 26 was not phosphorylated by these three protein kinases. Phosphopeptide mapping of connexin 32 demonstrated that cAMP-PK and PKC primarily phosphorylated a seryl residue in a peptide termed peptide 1. PKC also phosphorylated seryl residues in additional peptides. CA2+/CaM-PK II phosphorylated serine and to a lesser extent, threonine, at sites different from those phosphorylated by the other two protein kinases. A synthetic peptide PSRKGSGFGHRL-amine (residues 228-239 based on the deduced amino acid sequence of rat connexin 32) was phosphorylated by cAMP-PK and by PKC, with kinetic properties being similar to those for other physiological substrates phosphorylated by these enzymes. Ca2+/CaM-PK II did not phosphorylate the peptide. Phosphopeptide mapping and amino acid sequencing of the phosphorylated synthetic peptide indicated that Ser233 of connexin 32 was present in peptide 1 and was phosphorylated by cAMP-PK or by PKC. In hepatocytes labeled with [32P]orthophosphoric acid, treatment with forskolin or 20-deoxy-20-oxophorbol 12,13-dibutyrate (PDBt) resulted in increased 32P-incorporation into connexin 32. Phosphopeptide mapping and phosphoamino acid analysis showed that a seryl residue in peptide 1 was most prominently phosphorylated under basal conditions. Treatment with forskolin or PDBt stimulated the phosphorylation of peptide 1. PDBt treatment also increased the phosphorylation of seryl residues in several other peptides. PDBt did not affect the cAMP-PK activity in hepatocytes. It has previously been shown that phorbol ester reduces dye coupling in several cell types, however in rat hepatocytes, dye coupling was not reduced by treatment with PDBt. Thus, activation of PKC may have differential effects on junctional permeability in different cell types; one source of this variability may be differences in the sites of phosphorylation in different gap-junction proteins.     

The use of ubiquitin-peptide extensions as protein kinase substrates

Four ubiquitin-peptide extensions prepared as cloned products in E. coli were tested as casein kinase II substrates. Two extensions containing the sequence Ser-Glu-Glu-Glu-Glu-Glu were readily phosphorylated by partially purified rabbit reticulocyte casein kinase II. The other two fusion proteins, which lack a consensus phosphorylation site for casein kinase II, did not serve as substrates under identical reaction conditions. Native ubiquitin was not phosphorylated by reticulocyte casein kinase II, nor have we observed its phosphorylation in crude extracts from HeLa cells, mouse liver, or Xenopus eggs. Ubiquitin's apparent lack of phosphorylatable residues coupled with its remarkable heat stability and rapid migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels make the protein an attractive carrier for carboxyl-terminal peptides containing specific phosphorylation sites. Such ubiquitin extension proteins should prove valuable as protein kinase substrates.     

Structure and phosphorylation of eukaryotic initiation factor 2. Casein kinase 2 and protein kinase C phosphorylate distinct but adjacent sites in the beta-subunit

Eukaryotic initiation factor 2 (eIF-2) from rabbit reticulocytes can be phosphorylated on its beta-subunit by two different protein kinases, protein kinase C and casein kinase 2. Phosphorylation by these kinases is additive, suggesting that they phosphorylate different sites (serine residues) in eIF-2 beta. Two-dimensional peptide mapping of the phosphopeptides generated from labelled eIF-2 beta by digestion with trypsin, cyanogen bromide or Staphylococcus aureus V8 proteinase showed that protein kinase C and casein kinase 2 phosphorylated distinct and different sites in this protein. This conclusion was supported by the results of analysis of the phosphopeptides on reverse-phase chromatography. Analysis of the phosphopeptides derived from eIF-2 beta labelled by both kinases together strongly suggested that the sites labelled by protein kinase C and casein kinase 2 are adjacent in the primary sequence. These data are discussed in the light of the present understanding of the sequence specificity of the kinases. Rat liver eIF-2 beta was also found to be a substrate for protein kinase C and casein kinase 2, which were again shown to label different serine residues.     

Role of acidic residues as substrate determinants for casein kinase I

Sites phosphorylated by casein kinase I have been characterized by the presence of acidic amino acids NH2-terminal to the modified residue. Recently, phosphoserine was shown to be a particularly effective determinant for casein kinase I action when present in the motif -S(P)-X-X-S- (Flotow, H., Graves, P. R., Wang, A., Fiol, C. J., Roeske, R. W., and Roach, P. J. (1990) J. Biol. Chem. 265, 14264-14269). Nonetheless, nonphosphorylated substrates for casein kinase I are well documented. In this study, we examined the efficacy of Asp and Glu residues as determinants of casein kinase I action using synthetic peptide substrates. Peptides with runs of Asp residues in the motif Dn-X-X-S- were substrates for casein kinase I. Peptides with n = 3 or 4 were the most effective substrates, much better than n = 2. The peptide with n = 1, a single Asp residue, was a very poor substrate. A block of 4 Glu residues was a little less effective as a substrate determinant than 4 Asp residues in an otherwise identical peptide. The most effective substrate, with the motif -D-D-D-D-X-X-S-, was specific for casein kinase I and was not detectably phosphorylated by cyclic AMP-dependent protein kinase, casein kinase II, glycogen synthase kinase 3, or phosphorylase kinase and thus will be useful for the specific assay of casein kinase I. This peptide was nonetheless significantly worse as a substrate than peptides in which casein kinase I action was determined by phosphoserine in the -3 position. Still, the fact that Asp or Glu residues can specify a casein kinase I substrate suggests that acidic character has a role in substrate selection by this protein kinase.     

Phosphorylation of type II (beta) protein kinase C by casein kinase II.

Rat brain type II (beta) protein kinase C (PKC) was phosphorylated by rat lung casein kinase II (CK-II). Neither type I (gamma) nor type III (alpha) PKC was significantly phosphorylated by CK-II. CK-II incorporated 0.2-0.3 mol of phosphate into 1 mol of type II PKC. This phosphate was located at the single seryl residue (Ser-11) in the V1-variable region of the regulatory domain of the PKC molecule. A glutamic acid cluster was located at the carboxyl-terminal side of Ser-11, showing the consensus sequence for phosphorylation by CK-II. The velocity of this phosphorylation was enhanced by the addition of Ca2+, diolein, and phosphatidylserine, which are all required for the activation of PKC. Phosphorylation of casein or synthetic oligopeptides by CK-II was not affected by Ca2+, diolein, or phosphatidylserine. Available evidence suggests that CK-II phosphorylates preferentially the activated form of type II PKC. It remains unknown, however, whether this reaction has a physiological significance.     

A phosphatase resistant substrate for the assay of protein kinase C in crude tissue extracts.

Protein kinase C (PKC) is routinely assayed, after it is partially purified over DEAE-cellulose chromatography to eliminate any interfering protein kinases and phosphatases, by measuring the transfer of gamma-phosphate of [gamma-32P]ATP to H1 histone. Recently, it has been shown that a synthetic peptide, comprising residues 4-14 of myelin basic protein (MBP4-14), is a very selective PKC substrate which is not phosphorylated effectively by cyclic AMP-dependent protein kinase, casein kinase I and II, Ca2+/calmodulin dependent protein kinase II or phosphorylase kinase [Yasuda, I., Kishimoto, A., Tanaka, S-I., Tominaga, M., Sakurai, A. and Nishizuka, Y. (1990) BBRC 166, 1220-1227]. We report here that once MBP4-14 is phosphorylated, it is not dephosphorylated by okadaic acid-sensitive phosphatases (protein phosphatases 1, 2A and 3) or other protein phosphatases such as calcineurin and/or PP 2C present in hippocampal homogenates. Therefore, MBP4-14 can be used for PKC assay in crude extracts of neural tissue.     

Phosphorylation of maize RAB-17 protein by casein kinase 2

The maize gene RAB-17, which is responsive to abscisic acid, encodes a basic glycine-rich protein containing, in the middle part of its sequence, a cluster of 8 serine residues followed by a putative casein kinase 2-type substrate consensus sequence. This protein was found to be highly phosphorylated in vivo. Here, we show that RAB-17 protein is a real substrate for casein kinase 2. RAB-17 protein is phosphorylated in vitro by casein kinase 2 isolated from rat liver cytosol and from maize embryos. A maximum of 4 mol of phosphate were incorporated per mol of RAB-17 protein following incubation with casein kinase 2. Phosphopeptide mapping experiments show that the peptide phosphorylated by casein kinase 2 in vitro is identical to that derived from the protein phosphorylated in vivo. Purification by high performance liquid chromatography and partial sequencing of the phosphopeptide indicate that it corresponds to the region of the protein (residues 56-89) containing the cluster of serine residues. Our results indicate that RAB-17 is phosphorylated by casein kinase 2 or a kinase with a similar specificity and that phosphorylation takes place in the serine cluster region of the protein both in vitro and in vivo.     

Nucleolin (C23), a physiological substrate for casein kinase II

Nucleolin (C23), a 110 kDa phosphoprotein, which is mainly found in the nucleolus has been shown to be a physiological substrate for casein kinase II (CKII). Nucleolin was identified and characterized by immunodetection using an anti-nucleolin antibody. Phosphopeptide patterns from nucleolin phosphorylated by purified casein kinase II and of phosphorylated nucleolin which had been isolated from tumor cells grown in the presence of [32P]-o-phosphate, were identical. The partial tryptic digest revealed nine phosphopeptides. Nucleolin isolated from Krebs II mouse ascites cells was phosphorylated by purified casein kinase II with about two moles phosphate per one mole of nucleolin.     

Purification and characterization of a 400-kDa nonhistone chromatin protein that serves as an effective phosphate acceptor for casein kinase II from Ehrlich ascites tumor cells

A nonhistone chromatin protein (NHCP) has been purified to homogeneity from a 0.5 M NaCl extract of Ehrlich ascites tumor cell (EAT cell) nuclei as a phosphate acceptor for casein kinase II using ion-exchange column chromatographies and Sephacryl S300 gel filtration. The purified NHCP (approximate Mr = 400,000) was found to be a tetramer of an Mr = 98,000 polypeptide (pI = 6.9) and to have high contents of glycine (15%) and serine (11.6%). This protein (designated as 400-kDa NHCP) was highly phosphorylated by casein kinase II (Mr = 130,000), but not by histone kinase. Casein kinase II phosphorylated only seryl residues of the purified 400-kDa NHCP. The NHCP bound with DNA, but not with RNAs, and the DNA binding ability of the protein was reduced when it was phosphorylated by casein kinase II. Moreover, we found that (a) the 400-kDa NHCP is present in large quantities in malignant mouse cells, such as EAT, EL-4, and Meth-A cells, but only slightly in normal tissues and cells; (b) the protein level is rapidly increased when mouse lymphocytes are treated with recombinant interleukin 2 (T cell growth factor) or concanavalin A; and (c) the kinase responsible for the 400-kDa NHCP phosphorylation in the chromatin of various mouse cells is a casein kinase II. These experimental results suggest that the 400-kDa NHCP acts as an effective phosphate acceptor for casein kinase II at the chromatin level and that an increased phosphorylation of the protein by the kinase may be implicated in the progress of cell differentiation and proliferation.     

Phosphorylation of high mobility group protein 14 by casein kinase II

Phosphorylation of chromosomal high mobility group (HMG) protein 14 by casein kinase II has been characterized. Two mol of 32P are incorporated per mol of bovine HMG 14. Kinetic analysis provided evidence for two distinct sites with apparent Km values of 14.5 and 134 microM and respective Vmax values of 0.17 and 0.68 mumol/min/mg casein kinase II. Tryptic peptide mapping identified two phosphorylated products, each with phosphoserine. Amino acid composition and sequence analysis demonstrate that the major high affinity phosphorylation site for casein kinase II is serine 89. This sequence located at the carboxyl-terminal of HMG 14 contains the primary sequence determinants for casein kinase II. On the basis of reverse-phase high performance liquid chromatography and amino acid analysis, HMG 14, serine 99 represents the low affinity phosphorylation site.     

Nuclear protein phosphorylation in isolated nuclei from HeLa cells. Evidence that 32P incorporation from [gamma-32P]GTP is catalyzed by nuclear kinase II

A nuclear system for studying nuclear protein phosphorylation is characterized, using as phosphate donor either low levels of [gamma-32P]GTP, low levels of [gamma-32P]ATP, or low levels of labeled ATP plus excess unlabeled GTP. Since nuclear casein kinase II is the only described nuclear protein kinase to use GTP with high affinity, low levels of GTP should specifically assay this enzyme. ATP should measure all kinases, and ATP plus unlabeled GTP should measure all kinases except nuclear casein kinase II (ATP-specific kinases). The results are consistent with these predictions. In contrast with the ATP-specific activity, endogenous phosphorylation with GTP was enhanced by 100 mM NaCl, inhibited by heparin and quercetin, stimulated by polyamines, and did not use exogenous histone as substrate. The GTP- and ATP-specific kinases phosphorylated different subsets of about 20 endogenous polypeptides each. Addition of purified casein kinase II enhanced the GTP-supported phosphorylation of the identical proteins that were phosphorylated by endogenous kinase. These results support the hypothesis that activity measured with GTP is catalyzed by nuclear casein kinase II, though other minor kinases which can use GTP are not ruled out. Preliminary observations with this system suggest that the major nuclear kinases exist in an inhibited state in nuclei, and that the effects of polyamines on nuclear casein kinase II activity are substrate specific. This nuclear system is used to determine if the C-proteins of hnRNP particles, previously shown to be substrates for nuclear casein kinase II in isolated particles, is phosphorylated by GTP in intact nuclei. The results demonstrate that the C-proteins are effectively phosphorylated by GTP, but in addition they are phosphorylated by ATP-specific kinase activity.     

Phosphorylation of keratin intermediate filaments by protein kinase C, by calmodulin-dependent protein kinase and by cAMP-dependent protein kinase.

Keratins, constituent proteins of intermediate filaments of epithelial cells, are phosphoproteins containing phosphoserine and phosphothreonine. We examined the in vitro phosphorylation of keratin filaments by cAMP-dependent protein kinase, protein kinase C and Ca2+/calmodulin-dependent protein kinase II. When rat liver keratin filaments reconstituted by type I keratin 18 (molecular mass 47 kDa; acidic type) and type II keratin 8 (molecular mass 55 kDa; basic type) in a 1:1 ratio were used as substrates, all the protein kinases phosphorylated both of the constituent proteins to a significant rate and extent, and disassembly of the keratin filament structure occurred. Kinetic analysis suggested that all these protein kinases preferentially phosphorylate keratin 8, compared to keratin 18. The amino acid residues of keratins 8 and 18 phosphorylated by cAMP-dependent protein kinase or protein kinase C were almost exclusively serine, while those phosphorylated by Ca2+/calmodulin-dependent protein kinase II were serine and threonine. Peptide mapping analysis indicated that these protein kinases phosphorylate keratins 8 and 18 in a different manner. These observations gave the way for in vivo studies of the role of phosphorylation in the reorganization of keratin filaments.     

Phosphoserine in peptide substrates can specify casein kinase II action

Casein kinase II is a ubiquitous serine/threonine protein kinase which utilizes acidic amino acid residues as recognition determinants in its substrates, the motif -S/T-X-X-D/E- being particularly important. To test whether a phosphoserine residue can act as a substrate determinant, a peptide was synthesized, containing the sequence -S-X-X-S, which was not phosphorylated by casein kinase II. However, upon phosphorylation at the +3 position, the peptide became a substrate for casein kinase II. With another peptide, a positive influence of more distal phosphorylations was found. The results indicate the potential for casein kinase II to participate in hierarchal phosphorylation schemes.