Substrate For Casein Kinase Research Articles

Molecular surface sequence analysis of several E. coli enzymes and implications for existence of casein kinase-2 bacterial predecessor.

Casein kinase-2 (CK2) is known as pleiotropic eukaryotic protein kinase that phosphorylates significant number of cellular proteins. Not all functions of the protein were registered up to the present time. However, it is known that this Ser/Thr-specific kinase is involved in the cell cycle progression and is essentially required for the eukaryotic cell viability. Fully automated molecular surface analysis procedure for identification of functionally significant surface residues and sequences on the base of protein spatial structure was elaborated. Using the elaborated procedure, several E. coli enzymes spatial structures and sequences were investigated. It was found that most of the casein kinase 2 potential sites found in sequences of enzymes are accessible for modification. Four of the 5 structures studied have CK2 consensus sites that may definitely influence the activity of the enzyme upon phosphorylation. Some of the potential "CK2-sites" has amino acid contents characteristic for physiological substrates of casein kinase 2 in eukaryotes. The main point of the elaborated method and the structural evidence for existence of a putative casein kinase E. coli predecessor or a protein with similar kinase activity are discussed. Physiological, biochemical, structural and evolutionary aspects of the existence of the putative predecessor are considered.

A conserved acidic motif in the N-terminal domain of nitrate reductase is necessary for the inactivation of the enzyme in the dark by phosphorylation and 14-3-3 binding.

It has previously been shown that the N-terminal domain of tobacco (Nicotiana tabacum) nitrate reductase (NR) is involved in the inactivation of the enzyme by phosphorylation, which occurs in the dark (L. Nussaume, M. Vincentz, C. Meyer, J.P. Boutin, and M. Caboche [1995] Plant Cell 7: 611-621). The activity of a mutant NR protein lacking this N-terminal domain was no longer regulated by light-dark transitions. In this study smaller deletions were performed in the N-terminal domain of tobacco NR that removed protein motifs conserved among higher plant NRs. The resulting truncated NR-coding sequences were then fused to the cauliflower mosaic virus 35S RNA promoter and introduced in NR-deficient mutants of the closely related species Nicotiana plumbaginifolia. We found that the deletion of a conserved stretch of acidic residues led to an active NR protein that was more thermosensitive than the wild-type enzyme, but it was relatively insensitive to the inactivation by phosphorylation in the dark. Therefore, the removal of this acidic stretch seems to have the same effects on NR activation state as the deletion of the N-terminal domain. A hypothetical explanation for these observations is that a specific factor that impedes inactivation remains bound to the truncated enzyme. A synthetic peptide derived from this acidic protein motif was also found to be a good substrate for casein kinase II.

Age-dependent differences in glutamate-induced phosphorylation systems in rat hippocampal slices.

Glutamate receptor induced changes in the activity of different phosphorylation systems were measured in hippocampal slices from 12- and 56-day-old rats, by determining the endogenous phosphorylation of 2.5% perchloric acid (PCA) soluble proteins. We identified among these proteins an 85, 80 kDa and the tau protein as specific substrates for protein kinase A (PKA), MARCKS, and neurogranin as specific substrates for protein kinase C (PKC), and prostaglandin-D-synthase as substrate for casein kinase II (CKII). In addition, a 35 kDa protein was phosphorylated by calcium/calmodulin dependent kinase II and protein kinase C and a 21 kDa protein was a substrate for all investigated kinases. The basal endogenous phosphorylation of 2.5% PCA soluble proteins changed during development qualitatively and quantitatively. Thus, the phosphorylation degree of nearly all proteins declines during maturation. Activation of mGluR induced an increased phosphorylation of PKA, PKC, and CKII substrates in hippocampal slices from 12-day-old rats, but in slices of 56-day-old rats only PKA and to a lower extent PKC substrates were affected. In contrast, stimulation of NMDA receptors led to an enhancement of CKII and PKA dependent phosphorylation only in slices of young animals, whereas the endogenous phosphorylation of some proteins in adult slices was actually decreased. These data showing developmental changes in the coupling of metabotropic and ionotropic glutamate receptors to different phosphorylation systems are discussed in the light of altered physiological properties of the mature hippocampus.

Attenuation of drug-stimulated topoisomerase II-DNA cleavable complex formation in wild-type HL-60 cells treated with an intracellular calcium buffer is correlated with decreased cytotoxicity and site-specific hypophosphorylation of topoisomerase IIalpha.

Topoisomerase II (topo II), an essential enzyme for cell viability, is also the target for clinically important anti-neoplastic agents that stimulate topo II-mediated DNA scission. The role of alterations in topo IIalpha phosphorylation and its effect on drug-induced DNA damage and cytotoxicity were investigated. Following loading of HL-60 cells with the calcium buffer 1, 2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid tetra(acetoxymethyl) ester (BAPTA-AM), which abrogates intracellular Ca2+ transients, a significant decrease in etoposide (VP-16)- or amsacrine (m-AMSA)-stabilized topo II-DNA cleavable complex formation and a corresponding decrease in cytotoxicity was observed. In a cell-free system, nuclear extracts from BAPTA-AM-treated cells exhibited markedly less activity when assayed for VP-16-stabilized topo II-DNA complex formation, but not decatenation of kinetoplast DNA. In contrast, the loading of HL-60 cells with N,N,N', N'-tetrakis-(2-pyridyl)ethylenediamine (TPEN), which binds heavy metals without disturbing calcium or magnesium concentrations, did not significantly affect VP-16-stimulated topo II-DNA cleavable complex formation or cytotoxicity. In HL-60 cells the accumulation of BAPTA, but not TPEN, also led to the hypophosphorylation of topo IIalpha. Tryptic phosphopeptide mapping of topo IIalpha protein from HL-60 cells revealed: (a) eight major phosphorylation sites in untreated cells; (b) hypophosphorylation of two out of eight sites in BAPTA-AM-treated cells; and (c) hypophosphorylation of between two and four out of eight sites in topo II-poison-resistant HL-60 cells. The two hypophosphorylated sites present following BAPTA-AM treatment of wild-type cells were identical with the hypophosphorylated sites in the resistant cells, but were not the same as the sites that are substrates for casein kinase II [Wells, Addison, Fry, Ganapathi and Hickson (1994) J. Biol. Chem. 269, 29746-29751]. In summary, changes in intracellular Ca2+ transients that lead to the site-specific hypophosphorylation of topo IIalpha are possibly involved in regulating the DNA damage caused by and the cytotoxic potential of topo II poisons.

Regulation of gene expression by alternative polyadenylation and mRNA instability in hyperglycaemic mesangial cells.

We have used mRNA differential display to identify a novel high-glucose-regulated gene (HGRG-14) in human mesangial cells cultured for up to 21 days in 30 mM d-glucose. The mRNA of HGRG-14 seems to be regulated post-transcriptionally and encodes a small polypeptide of molecular mass 13 kDa. The native protein occurs as a dimer. The recombinant protein is a substrate for casein kinase II kinase. At high glucose concentrations, HGRG-14 protein levels decrease. This correlates with the appearance of a long form of HGRG-14 mRNA under high-glucose conditions. This form has a long 3' untranslated region containing several ATTTA RNA-destabilizing sequences and has a short half-life. A truncated, more stable mRNA that lacks the long 3' untranslated region is produced at 4 mM d-glucose. The switch from the truncated to the long-form transcript is detected within 2 h of exposure to 30 mM d-glucose, indicating that hyperglycaemic conditions have an acute effect on HGRG-14 mRNA processing.

Kell and Kx, Two Disulfide-Linked Proteins of the Human Erythrocyte Membrane Are Phosphorylated in Vivo

Kell and Kx are two quantitatively minor proteins from the human erythrocyte membrane which carry blood groups antigens and are thought to be a metalloprotease and a membrane transporter, respectively. In the red cell membrane, these proteins form a complex stabilized by disulfide bond(s). Phosphorylation status of these proteins was studied, in the presence or absence of effectors of several kinases, either on intact cells incubated with [ 32P]-orthophosphate or on ghosts incubated with [γ- 32P]ATP. Purification of Kell-Kx complex, by immunochromatography on an immobilized human monoclonal antibody of Kell blood group specificity allowed to establish that (i) neither protein is phosphorylated on tyrosine; (ii) the Kell protein is a putative substrate for Casein Kinase II (CKII) and Casein Kinase I (CKI) but not for protein kinase C (PKC), whereas Kx protein is phosphorylated by CKII and PKC but not by CKI; (iii) Protein Kinase A neither phosphorylates the Kell nor the Kx proteins.

Casein Kinase II Stabilizes the Activity of Human Topoisomerase IIα in a Phosphorylation-independent Manner

Previous reports have indicated that topoisomerase II (topo II) co-purifies with and is a substrate for casein kinase II. We have carried out a detailed study of the effect that purified casein kinase II has on the activity of purified recombinant human topo IIalpha. Co-incubation of topo IIalpha and casein kinase II led to an apparent activation of the topo IIalpha; however, in experiments in which topo IIalpha was preincubated at 37 degrees C with or without native casein kinase II prior to assaying for decatenation activity, it emerged that the kinase was exerting its "activating" function via a decrease in the rate of topo IIalpha enzyme inactivation during the incubation period. This stabilization of topo IIalpha by casein kinase II was ATP-independent and was observed in both mutated and truncated derivatives of topo IIalpha lacking the major casein kinase II phospho-acceptor sites, indicating the lack of a requirement for phosphorylation. Consistent with a nonenzymatic role for casein kinase II, stoichiometric quantities of kinase were required for topo IIalpha stabilization. These data indicate that casein kinase II plays a significant role in regulating human topo IIalpha protein action via stabilization against thermal inactivation.

Purification, Characterization, andin VitroPhosphorylation of the Neuron-Specific Membrane-Associated Protein SCG10

SCG10 is a neuron-specific, developmentally regulated protein which is highly enriched in growth cones. Sequence homology indicates that it is related to the phosphoprotein stathmin or Op18, anin vitroandin vivosubstrate for several serine/threonine kinases which are involved in a variety of signaling pathways. As a first step to examine the biochemical properties of SCG10, the protein was expressed inEscherichia coliand purified to apparent homogeneity. The purified protein was used inin vitrophosphorylation assays. SCG10 was phosphorylated by MAP kinase, cAMP-dependent protein kinase, cGMP-dependent protein kinase, p34cdc2kinase, DNA-dependent protein kinase, Ca2+/calmodulin kinase II, and casein kinase II. The protein was not a substrate for casein kinase I and protein kinase C. SCG10 was phosphorylated by src tyrosine kinase, which demonstrates that the protein can be phosphorylatedin vitroon a tyrosine residue. Our data suggest that SCG10 is a phosphoprotein which might be involved in signal transduction in neurons.

A novel, calcium-inhibitable casein kinase in Paramecium cells

This is the first identification of a Ca 2+-inhibitable casein kinase (CPK) which we have isolated from the 100 000× g supernatant of Paramecium cell homogenates. The 1000-fold enriched CPK activity depends on millimolar Mg 2+ and is inhibited by low concentrations of heparin or by ≥100 μM Ca 2+. Enzyme activity is stimulated by polylysine or polyarginine with either casein or with specific casein kinase-2 (CK-2) peptide substrates (RRRDDDSDDD and RREEETEEE). The enzymic properties are similar with GTP instead of ATP. CPK does not undergo autophosphorylation. In gel kinase assays, enzyme activity is associated with a 36 kDa band. Calmodulin as another characteristic substrate for mammalian CK-2 has not been phosphorylated by this protein kinase. Besides casein, CPK phosphorylates in vitro the catalytic subunit of bovine brain calcineurin (CaN), a typical substrate of type 1 mammalian casein kinase (CK-1) in vitro. Again this phosphorylation is significantly reduced by Ca 2+. Thus, CPK combines aspects of different casein kinases, but it is clearly different from any type known by its Ca 2+ inhibition. Since CPK also phosphorylates the exocytosis-sensitive phosphoprotein, PP63, in Paramecium, which is known to be dephosphorylated by CaN, an antagonistic Ca 2+-effect during phosphorylation/dephosphorylation cycles may be relevant for exocytosis regulation.

An abundant nucleolar phosphoprotein is associated with ribosomal DNA in Tetrahymena macronuclei.

An abundant 52-kDa phosphoprotein was identified and characterized from macronuclei of the ciliated protozoan Tetrahymena thermophila. Immunoblot analyses combined with light and electron microscopic immunocytochemistry demonstrate that this polypeptide, termed Nopp52, is enriched in the nucleoli of transcriptionally active macronuclei and missing altogether from transcriptionally inert micronuclei. The cDNA sequence encoding Nopp52 predicts a polypeptide whose amino-terminal half consists of multiple acidic/serine-rich regions alternating with basic/proline-rich regions. Multiple serines located in these acidic stretches lie within casein kinase II consensus motifs, and Nopp52 is an excellent substrate for casein kinase II in vitro. The carboxyl-terminal half of Nopp52 contains two RNA recognition motifs and an extreme carboxyl-terminal domain rich in glycine, arginine, and phenylalanine, motifs common in many RNA processing proteins. A similar combination and order of motifs is found in vertebrate nucleolin and yeast NSR1, suggesting that Nopp52 is a member of a family of related nucleolar proteins. NSR1 and nucleolin have been implicated in transcriptional regulation of rDNA and rRNA processing. Consistent with a role in ribosomal gene metabolism, rDNA and Nopp52 colocalize in situ, as well as by cross-linking and immunoprecipitation experiments, demonstrating an association between Nopp52 and rDNA in vivo.

MFP1, a novel plant filament-like protein with affinity for matrix attachment region DNA.

The interaction of chromatin with the nuclear matrix via matrix attachment regions (MARs) on the DNA is considered to be of fundamental importance for higher order chromatin organization and regulation of gene expression. Here, we report a novel nuclear matrix-localized MAR DNA binding protein, designated MAR binding filament-like protein 1 (MFP1), from tomato. In contrast to the few animal MAR DNA binding proteins thus far identified, MFP1 contains a predicted N-terminal transmembrane domain and a long filament-like alpha-helical domain that is similar to diverse nuclear and cytoplasmic filament proteins from animals and yeast. DNA binding assays established that MFP1 can discriminate between animal and plant MAR DNAs and non-MAR DNA fragments of similar size and AT content. Deletion mutants of MFP1 revealed a novel, discrete DNA binding domain near the C terminus of the protein. MFP1 is an in vitro substrate for casein kinase II, a nuclear matrix-associated protein kinase. Its structure, MAR DNA binding activity, and nuclear matrix localization suggest that MFP1 is likely to participate in nuclear architecture by connecting chromatin with the nuclear matrix and potentially with the nuclear envelope.

Phosphorylation of synaptic vesicle proteins: modulation of the alpha SNAP interaction with the core complex.

We analyzed whether synaptic membrane trafficking proteins are substrates for casein kinase II, calcium/calmodulin-dependent protein kinase II, and cAMP-dependent protein kinase (PKA), three kinases implicated in the modulation of synaptic transmission. Each kinase phosphorylates a specific set of the vesicle proteins syntaxin 1A, N-ethylmaleimide-sensitive factor (NSF), vesicle-associated membrane protein (VAMP), synaptosome-associated 25-kDa protein (SNAP-25), n-sec1, alpha soluble NSF attachment protein (alpha SNAP), and synaptotagmin. VAMP is phosphorylated by calcium/calmodulin-dependent protein kinase II on serine 61. alpha SNAP is phosphorylated by PKA; however, the beta SNAP isoform is phosphorylated only 20% as efficiently. alpha SNAP phosphorylated by PKA binds to the core docking and fusion complex 10 times weaker than the dephosphorylated form. These studies provide a first glimpse at regulatory events that may be important in modulating neurotransmitter release during learning and memory.

Evidence for a phosphorylation site in cytomegalovirus glycoprotein gB.

As part of our vaccine program, we have purified a recombinant form of human cytomegalovirus glycoprotein B that is able to induce high titers of virus-neutralizing antibodies. The isolated protein was found to be phosphorylated at a serine residue in position -7 from the C terminus of the protein. The corresponding synthetic peptide, HLKDSDEEENV, was an efficient in vitro substrate of casein kinase II.

Studies on the phosphorylation of a M(r) 25,000 protein, a putative protein phosphatase 2A modulator, by casein kinase I, and analysis of multiple endogenous phosphates.

A M(r) 25,000 protein, which was isolated from the cytosolic fraction of Xenopus laevis oocytes, is a newly identified substrate for casein kinase II and protein kinase C [Hashimoto et al. (1995) J. Biochem. 118, 453-460], and was recently shown to have the ability to modulate protein phosphatase 2A activity [Hashimoto et al. (1996) J. Biochem. 119, 626-632]. Acid phosphatase treatment of the protein shifted its electrophoretic mobility from 25 to 20 kDa on SDS-PAGE. The content of alkali-labile phosphate bound covalently to the protein was 53 mol per mol of M(r) 25,000 protein. Amino acid composition analysis revealed that there are 50 serine residues and 6 threonine residues per mol of this protein. Therefore, this M(r) 25,000 protein seems to be highly phosphorylated in vivo. The M(r) 25,000 protein, once partially dephosphorylated by acid phosphatase, served as an efficient substrate for casein kinase I and casein kinase II. When entirely dephosphorylated, the M(r) 25,000 protein was used as a substrate, the rate of phosphorylation with both casein kinases being decreased. This behavior of casein kinases toward the M(r) 25,000 protein reflects the possible mechanism of multisite phosphorylation in which the introduction of a phosphate group facilitates sequential phosphorylation.

Evidence that casein kinase 2 phosphorylates hepatic microsomal calcium-binding proteins 1 and 2 but not 3.

We have extensively purified three of the hepatic microsomal intralumenal Ca2+-binding proteins, CBP1, CBP2, and CBP3, which were originally described by Van et al. [(1989) J. Biol. Chem. 264, 17494-17501]. These apparently homogeneous preparations showed only single 45Ca2+ binding bands. On the basis of the peptide sequence, CBP2 was found to be highly homologous with the previously described protein ERp72. Similarly, CBP3 was identical to calreticulin and CBP1 had some homology to calmodulin. Contrary to the report of Van et al. (1989), we found that CBP2 had little thiol:protein disulfide oxidoreductase activity. Of the three purified preparations, only CBP2 exhibited apparent intrinsic protein kinase activity. This activity was found to be due to contamination of the CBP2 preparation by an extremely low concentration of tightly bound casein kinase 2 (CK2). In line with this observation, the phosphorylation was inhibited by heparin, removed by antibody to CK2, and stimulated by spermine. Furthermore, CBP2 was readily phosphorylated in vitro by added CK2 but only slowly phosphorylated by several other protein kinases. Thus, the persistence of CK2 in a highly purified preparation of CBP2 along with several other lines of evidence presented in this study might suggest that the protein CBP2 is a physiologically relevant substrate for CK2. Furthermore, these data suggest that CK2 might be localized in the lumen of the endoplasmic reticulum and that the phosphorylation of CBP2 in the lumen may play a role in the chaperone activity attributed to this protein.

Purification and phosphorylation of a M(r) 25,000 protein, an effective phosphate acceptor for casein kinase II and protein kinase C, detected in the cytosolic fraction of Xenopus laevis oocytes.

A common and effective phosphate acceptor protein for casein kinase II and Ca(2+)-phospholipid-dependent protein kinase (protein kinase C) was purified to near homogeneity from the cytosolic fraction of Xenopus laevis oocytes. Its molecular mass was estimated to be approximately 25,000 by SDS-polyacrylamide slab gel electrophoresis and gel filtration analyses. About 1 and 2 mol of phosphate were incorporated per mol of this protein with casein kinase II and protein kinase C, respectively, and the phosphorylated amino acid was identified as serine irrespective of the protein kinase employed. The Km values were calculated to be 1 and 0.5 microM for this M(r) 25,000 protein with casein kinase II and protein kinase C, respectively. However, this protein was a relatively poor substrate for casein kinase I and did not serve as one for cAMP-dependent protein kinase. The amino acid sequence of its amino-terminal region suggests that this protein is a newly identified substrate for these two protein serine/threonine kinases.

Phosphorylation of DARPP-32, a Dopamine- and cAMP-regulated Phosphoprotein, by Casein Kinase I in Vitro and in Vivo

DARPP-32 (dopamine- and cAMP-regulated phosphoprotein, M(r) = 32,000) is a potent inhibitor of protein phosphatase-1 when it is phosphorylated on Thr-34 by cAMP-dependent protein kinase. DARPP-32 is highly enriched in some specific cell populations such as striatonigral neurons and choroid plexus epithelial cells. Here we show that recombinant rat DARPP-32 is phosphorylated by casein kinase I on seryl residues to a stoichiometry of approximately 2 mol of phosphate/mol of protein. DARPP-32 is one of the best known substrates for casein kinase I (Km = 3.4 +/- 0.3 microM), whereas the homologous phosphatase-1 inhibitor, inhibitor-1, is not. Phosphorylation of DARPP-32 by casein kinase I does not alter its ability to inhibit protein phosphatase-1. Residues phosphorylated by casein kinase I were identified as Ser-137 and Ser-189 by site-directed mutagenesis and by protein sequencing. Ser-137 and the preceding stretch of 16-18 acidic residues are conserved in DARPP-32 among all species examined, whereas Ser-189 is not. Phosphorylation of Ser-137 induces an unusual increase in DARPP-32 electrophoretic mobility in polyacrylamide gels in the presence of SDS. In striatonigral neurons, DARPP-32 is phosphorylated on Ser-137 and the stoichiometry of phosphorylation on this residue in vivo appears to be higher in the substantia nigra (axon terminals) than in the striatum (soma and dendrites). These results indicate that casein kinase I is highly active in striatonigral neurons in which it may play important roles, including in protein phosphatase-1 modulation via phosphorylation of DARPP-32.

Serine 1524 is a major site of phosphorylation on human topoisomerase II alpha protein in vivo and is a substrate for casein kinase II in vitro.

Topoisomerase II protein is essential for cell proliferation and is known to exist as a phosphoprotein in cells from both lower and higher eukaryotic species. In this paper, we have investigated the phosphorylation of the alpha isozyme of human topoisomerase II. The topoisomerase II alpha protein was phosphorylated predominantly on serine residues in the human tumor cell lines HeLa and NSCLC-3. Two-dimensional tryptic phosphopeptide mapping studies revealed several sites of phosphorylation in vivo, including a major site that was common to topoisomerase II alpha protein from both HeLa and NSCLC-3 cells. To identify sites of phosphorylation, the regulatory C-terminal domain of human topoisomerase II alpha protein was overexpressed in Escherichia coli as a hexahistidine-tagged fusion protein and purified by nickel chelate chromatography. Tryptic phosphopeptide mapping revealed that casein kinase II phosphorylated the C-terminal domain primarily on 2 serine residues in vitro, which were shown to be sites of modification in vivo. Site-directed mutagenesis studies identified these casein kinase II-specific phosphorylation sites as serine 1524 and serine 1376.

Phosphorylation of caldesmon by smooth-muscle casein kinase II.

A caldesmon kinase activity was partially purified from an extract of chicken gizzard smooth muscle by sequential chromatography on columns of DEAE-Sephacel, MonoQ and Superose 12. This kinase was identified as casein kinase II by Western blotting using peptide-directed antibodies raised against the alpha, alpha' and beta subunits of human casein kinase II; the smooth muscle enzyme consisted of similar subunits of M(r) 43,000 (alpha), 39,000 (alpha'), and 27,000 (beta). Phosphorylation of caldesmon and casein by smooth muscle casein kinase II was optimal at approximately 0.1 M NaCl, did not require second messengers, and was inhibited by heparin. The kinase utilized either GTP or ATP as a substrate. Caldesmon was phosphorylated to approximately 1 mol Pi mol-1 caldesmon by smooth muscle casein kinase II with a Km for caldesmon of 4.9 microM. Two-dimensional thin-layer electrophoresis indicated phosphate incorporation into both serine and threonine. All the incorporated phosphate was recovered in the N-terminal peptide (residues 1-152) generated by cleavage at cysteine 153 with 2-nitro-5-thiocyanobenzoic acid. Purification of tryptic phosphopeptides and N-terminal sequencing revealed two principal sites of phosphorylation: serine 73 and threonine 83. The following four synthetic peptides corresponding to this domain of caldesmon were examined as substrates of casein kinase II: A = RRREVNAQNSVAEEE; B = AQNSVAEEE; C = RSTDDEAA; D = SVAEEETKRSTDDE. Interestingly, only peptides C and D were phosphorylated and both only at threonine. Phosphorylation of intact caldesmon did not affect the pattern of chymotryptic digestion suggesting that it does not induce a significant conformational change in the protein substrate. Phosphorylation also had no effect on the binding of caldesmon to actin or on the caldesmon-mediated inhibition of actomyosin MgATPase activity. However, phosphorylation completely abolished the interaction of caldesmon with immobilized smooth muscle myosin. These results are consistent with the localization of the myosin-binding domain near the N-terminus of caldesmon and of the actin-binding domain near the opposite end of the elongated molecule. Casein kinase II may therefore play a role in regulating caldesmon-myosin interaction and the ability of caldesmon to cross-link actin and myosin filaments in smooth muscle.

D-Amino Acid Residues as Substrate Specificity Determinants for Casein Kinase II

A set of diastereomeric peptides RRRDDDSDDD each with a corresponding D-amino acid residue successively in every position (except the arginines) was tested as substrates for casein kinase II. It was found that the D-serine containing peptide was not detectably phosphorylated. The replacements at the positions +1, −1 and +2 decreased the k II values more than 60, 30 and 20 times, respectively. The effect of the L/D replacements decreased with increased distance from the serine. The D-amino acid scan used herein seems to be a helpful complementary tool for studies of substrate specificity determinants for different protein kinases.