Phosphorylation of src-phosphopeptides by casein kinases-1 and -2: favourable effect of phosphotyrosine.

The synthetic phosphotyrosyl tridecapeptide H-Arg-Leu-Ile-Glu-Asp-Asn-Glu-Tyr(P)-Thr-Ala-Arg-Gln-Gly-OH, reproducing a major phosphoacceptor site of protein tyrosine kinases of the src-family, can be phosphorylated at Thr-9 by both casein kinases -1 and -2. Its shorter derivative H-Asn-Glu-Tyr(P)-Thr-Ala-OH is not affected by casein kinase-1 while representing a substrate as good as the tridecapeptide for casein kinase-2. The unphosphorylated analogue H-Asn-Glu-Tyr-Thr-Ala-OH, however, is a much poorer substrate, and no significant phosphorylation could be observed of its O-methyl ether derivative H-Asn-Glu-Tyr(Me)-Thr-Ala-OMe. These data on one side corroborate the concept that casein kinase-1 recognizes residues located on the C-terminal edge of acidic stretches, providing, on the other, the evidence that phosphotyrosyl side chains can act as specificity determinants for casein kinase-2.     

Phosphorylation of the phosphatase modulator subunit (inhibitor-2) by casein kinase-1. Identification of the phosphorylation sites.

The isolated modulator subunit of the inactive protein phosphatase-1 is phosphorylated in vitro by casein kinase-1 at two different sites: Ser-86 and Ser-174. The Ser-86 site is a common target for casein kinase-1 and casein kinase-2, but is preferentially phosphorylated by the former enzyme. The Ser-174 site seems to be specific for casein kinase-1, and is phosphorylated at a slower rate. These results give a new insight into the in vitro phosphorylation pattern of the modulator subunit of the phosphatase and provides additional data on the specificity of casein kinase-1.     

The phosphorylation of nucleoplasmin by casein kinase-2 is resistant to heparin inhibition

Highly purified preparations of casein kinase-2 from the nuclei of Xenopus laevis oocytes and from calf thymus can phosphorylate in vitro purified nucleoplasmin from X. laevis oocytes and eggs. The phosphorylation of nucleoplasmin by both kinase preparations is quite insensitive to heparin in contrast with casein phosphorylation which is completely abolished by heparin concentrations above 10 micrograms/ml. However, the phosphorylation of nucleoplasmin and casein are inhibited in a very similar fashion by 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), a well characterized specific inhibitor of casein kinase-2. Similarly, nucleoplasmin phosphorylation by the oocyte enzyme can be stimulated several-fold by spermine, another characteristic of this enzyme. These findings indicate that the phosphorylation of nucleoplasmin by purified casein kinase-2, while showing typical response to DRB and spermine, exhibits anomalous behavior in its resistance to heparin inhibition. It is possible that the large clusters of acidic amino acids in nucleoplasmin permit this substrate to interact with the enzyme more efficiently than other protein substrates. Heparin is generally considered a potent and specific inhibitor of casein kinase-2. This study, however, questions the validity of utilizing heparin inhibition as a criterion for casein kinase-2 involvement.     

Synthetic fragments of beta-casein as model substrates for liver and mammary gland casein kinases

The octapeptide Glu-Ser-Leu-Ser-Ser-Ser-Glu-Glu, corresponding to the 14-21 sequence of bovine beta-casein A2 and 11 shorter and/or modified derivatives were synthesized and used as model substrates for three casein kinases: rat liver casein kinases 2 and 1 and a casein kinase isolated from the golgi-enriched fraction of lactating mammary gland (GEF-casein kinase). Casein kinase-2 readily phosphorylates the octapeptide at its Ser-4 residue with a Vmax value comparable to those obtained with protein substrates and Km values of 85 microM and 11 microM in the absence and presence of polylysine, respectively. These are the most favourable kinetic parameters reported so far with peptide substrates of casein kinase-2. Stepwise shortening of the octapeptide from its N terminus promotes both a gradual decrease of Vmax and an increase of Km, this being especially dramatic in passing from the hexapeptide Leu-Ser-Ser-Ser-Glu-Glu (Km 210 microM) to the pentapeptide Ser-Ser-Ser-Glu-Glu (Km 2630 microM). The tetrapeptide Ser-Ser-Glu-Glu is the shortest derivative still phosphorylated by casein kinase-2, albeit very slowly, and the tripeptides Ser-Glu-Glu and Glu-Leu-Ser were not substrates at all. Furthermore, the pentapeptide Ser-Ser-Ser-Glu-Glu was found to be a better substrate than Ser-Ser-Ala-Glu-Glu, Ser-Ala-Ser-Glu-Glu and Ser-Ala-Ala-Glu-Glu by virtue of its lower Km value. These data, while confirming that the motif Ser-Xaa-Xaa-Glu is specifically recognized by casein kinase-2, strongly suggest that additional local structural features can improve the phosphorylation efficiency of serine-containing peptides which are devoid of the large acidic clusters recurrent in many phosphorylation sites of casein kinase 2. In particular, predictive structural analysis as well as NMR and C18 reverse-phase HPLC elution profile data support the hypothesis that a beta-turn conformation is responsible for the remarkable suitability of the octapeptide Glu-Ser-Leu-Ser-Ser-Ser-Glu-Glu and some of its shorter derivatives to phosphorylation mediated by casein kinase-2. While neither the peptide Glu-Ser-Leu-Ser-Ser-Ser-Glu-Glu nor any of its derivatives were affected by casein kinase-1, a rapid phosphorylation of the octapeptide by GEF-casein kinase at Ser-5 (not Ser-4) was obtained.(ABSTRACT TRUNCATED AT 400 WORDS)     

Analysis of sites phosphorylated on acetyl-CoA carboxylase in response to insulin in isolated adipocytes. Comparison with sites phosphorylated by casein kinase-2 and the calmodulin-dependent multiprotein kinase

We have examined the sites phosphorylated on acetyl-CoA carboxylase in response to insulin in isolated adipocytes. Two tryptic peptides derived from the enzyme become more radioactive after treatment of 32P-labelled cells with insulin. One of these (T4a) accounts for a large part of the total increase in phosphate observed after insulin treatment, and comigrates with the peptide containing the sites phosphorylated in vitro by casein kinase-2. The other may correspond to the 'I' site peptide originally described by Brownsey and Denton in 1982: labelling of this peptide is stimulated at least threefold by insulin treatment, but it is a minor phosphopeptide and, even after insulin treatment, accounts for only about 2.5% of the enzyme-bound phosphate (equivalent to less than 0.1 mol phosphate/mol 240-kDa subunit). Two other major tryptic phosphopeptides (T1 and T4b) labelled in adipocytes do not change significantly in response to insulin, and comigrate with peptides containing sites phosphorylated in vitro by cyclic-AMP-dependent protein kinase and calmodulin-dependent multiprotein kinase respectively. We have sequenced peptides T4a and T4b from acetyl-CoA carboxylase derived from control and insulin-treated adipocytes, and also after phosphorylation in vitro with casein kinase-2 and the calmodulin-dependent multiprotein kinase. The results show that T4a and T4b are forms of the same peptide containing phosphate groups on different serine residues: Phe-Ile-Ile-Gly-Ser4-Val-Ser5-Gln-Asp-Asn-Ser6-Glu-Asp -Glu-Ile-Ser-Asn-Leu-. Site 5 was phosphorylated by the calmodulin-dependent protein kinase and site 6 by casein kinase-2. Migration in the T4a position was exclusively associated with phosphorylation in site 6, irrespective of the presence of phosphate in sites 4 and 5. Sites 5 and 6 were partially phosphorylated in control adipocytes, and there were also small amounts of phosphate in site 4. On stimulation with insulin, phosphorylation appeared to occur primarily at site 6, thus accounting for the increase in 32P-labelling of T4a. We were unable to isolate sufficient quantities of the other insulin-sensitive peptide to determine its sequence. Our results are consistent with the idea that insulin activates either casein kinase-2, or a protein kinase which has the same specificity as casein kinase-2. The function of this modification is not clear, since phosphorylation by casein kinase-2 has no direct effect on acetyl-CoA carboxylase activity.     

Phosphorylation of phosvitin by casein kinase-2 provides the evidence that phosphoserines can replace carboxylic amino acids as specificity determinants

The consensus sequence of casein kinase-2 consists of a serine (threonine) followed by a cluster of glutamic and/or aspartic acids, the one at position +3 playing an especially crucial role (Marin et al., (1986) Eur. J. Biochem. 160, 239-244 and Kuenzel et al. (1987) J. Biol. Chem. 262, 9136-9140). None of the 123 serines of the main phosvitin component (34 kDa) fulfils such a requirement (Byrne et al. (1984) Biochemistry 23, 4275-4279), rather, most of them are clustered into stretches of up to 14 entirely phosphorylated residues. Three out of the four threonines lie close to the N-terminal side of such phosphoseryl blocks. Here we show that native 34 kDa phosvitin is a poor substrate of casein kinase-2, its radiolabeling occurring mostly at threonine residue(s); a very slight (1%) previous dephosphorylation with acid phosphatase converts phosvitin into an excellent substrate for casein kinase-2, its phosphorylation occurring almost exclusively at serine residues. Extensive dephosphorylation however (greater than 40%) reduces the phosphorylation efficiency of casein kinase-2. These results show that phosphoserine residues can replace carboxylic residues as specificity determinants for casein kinase-2.     

Phosphorylation of troponin and myosin light chain by cAMP-independent casein kinase-2 from rabbit skeletal muscle

Casein kinase-2 from rabbit skeletal muscle was found to phosphorylate, in addition to glycogen synthase, troponin from skeletal muscle, and myosin light chain from smooth muscle. Troponin T and the 20,000 M_r myosin light chain are phosphorylated by casein kinase-2 at much greater rates than glycogen synthase. The V values for the phosphorylation of troponin and myosin light chain are nearly an order of magnitude greater than that of glycogen synthase; however, the K_m values for these two substrates are greater than that for glycogen synthase. The kinase activities with the various protein substrates are stimulated approximately three- and fivefold by 5 mm spermidine and 3 mm spermine, respectively. Heparin is a potent inhibitor of the kinase when casein, glycogen synthase, or myosin light chain is the substrate. However, with troponin as substrate the kinase is relatively insensitive to inhibition by heparin. The amount of heparin required for 50% inhibition with troponin as substrate is at least 10 times greater than with casein as substrate. The phosphorylation of troponin by casein kinase-2 results in the incorporation of phosphate into two major tryptic peptides, which are different from those phosphorylated by casein kinase-1. The site in myosin light chain phosphorylated by casein kinase-2 is different from that phosphorylated by myosin light chain kinase.     

Crystal structure of a conformation-selective casein kinase-1 inhibitor

Members of the casein kinase-1 family of protein kinases play an essential role in cell regulation and disease pathogenesis. Unlike most protein kinases, they appear to function as constitutively active enzymes. As a result, selective pharmacological inhibitors can play an important role in dissection of casein kinase-1-dependent processes. To address this need, new small molecule inhibitors of casein kinase-1 acting through ATP-competitive and ATP-noncompetitive mechanisms were isolated on the basis of in vitro screening. Here we report the crystal structure of 3-[(2,4,6-trimethoxyphenyl) methylidenyl]-indolin-2-one (IC261), an ATP-competitive inhibitor with differential activity among casein kinase-1 isoforms, in complex with the catalytic domain of fission yeast casein kinase-1 refined to a crystallographic R-factor of 22.4% at 2.8 A resolution. The structure reveals that IC261 stabilizes casein kinase-1 in a conformation midway between nucleotide substrate liganded and nonliganded conformations. We propose that adoption of this conformation by casein kinase-1 family members stabilizes a delocalized network of side chain interactions and results in a decreased dissociation rate of inhibitor.     

Synthetic peptides including acidic clusters as substrates of yeast casein kinase-2

The synthesis is reported of a series of glutamyl peptide analogs of the model substrate H-Ser-Glu-Glu-Glu-Glu-Glu-OH of casein kinase-2 (CK-2). A convenient HPLC method for the separation of slightly different acidic peptides is also reported. The site specificity of yeast casein kinase-2 (Y-CK2) is examined with the aid of synthesized peptide substrates.     

Phosphorylation and inactivation of rabbit skeletal muscle glycogen synthase: distinction between kinase Fa-, phosphorylase kinase-, and glycogen synthase (casein) kinase-1-catalyzed reactions

Rabbit skeletal muscle glycogen synthase was phosphorylated by kinase Fa, phosphorylase kinase, and cAMP-independent synthase (casein) kinase-1 to determine the differences among these kinase-catalyzed reactions. The stoichiometry of phosphate incorporation, the extent of inactivation, and the sites of phosphorylation were compared. Synthase (casein) kinase-1 catalyzes the highest level of synthase phosphorylation (4 mol/subunit) and inactivation (reduction of the activity ratio to below 0.05). The sites, defined by characteristic tryptic peptides, phosphorylated by synthase (casein) kinase-1 are distinguishable from those by kinase Fa and phosphorylase kinase. In addition, synthase (casein) kinase-1, unlike kinase Fa, does not activate ATP X Mg2+-dependent protein phosphatase. These results demonstrate that synthase (casein) kinase-1 is a distinct glycogen synthase kinase.     

Identification of the sites on rabbit skeletal muscle protein phosphatase inhibitor-2 phosphorylated by casein kinase-II

Inhibitor-2 was phosphorylated by casein kinase-II in vitro at a rate similar to that of glycogen synthase, a physiological substrate of this protein kinase. The major phosphorylation sites were identified as serines-86, -120 and -121, the peptide containing serines-120 and -121 being labelled about 2.5-fold more rapidly than that containing serine-86. The 13 residues C-terminal to serine-121 (SGEEDSDLSPEERE) contain seven acidic amino acids, while the six residues following serine-86 (SDTETTE) contain three. These results are consistent with the known specificity requirements of casein kinase-II. The three serines are C-terminal to the threonine (residue 72) whose phosphorylation by glycogen synthase kinase-3 is potentiated by prior phosphorylation with casein kinase-II. This reinforces the view that a C-terminal phosphoserine residue is important for the specificity of glycogen synthase kinase-3. Identification of the residues phosphorylated by casein kinase-II will facilitate further studies on the in vivo phosphorylation state of inhibitor-2.     

Insulin receptor serine kinase activation by casein kinase 2 and a membrane tyrosine kinase

The insulin receptor (IR) tyrosine kinase can apparently directly phosphorylate and activate one or more serine kinases. The identities of such serine kinases and their modes of activation are still unclear. We have described a serine kinase (here designated insulin receptor serine (IRS) kinase) from rat liver membranes that co-purifies with IR on wheat germ agglutinin-agarose. The kinase was activated after phosphorylation of the membrane glycoproteins by casein kinase-1, casein kinase-2, or casein kinase-3 (Biochem Biophys Res Commun 171:75–83, 1990). In this study, IRS kinase was further characterized. The presence of vanadate or phosphotyrosine in reaction mixtures was required for activation to be observed. Phosphoserine and phosphothreonine are only about 25% as effective as phosphotyrosine, whereas sodium fluoride and molybdate were ineffective in supporting activation. Vanadate and phosphotyrosine support IRS kinase activation by apparently inhibiting phosphotyrosine protein phosphatases present among the membrane glycoproteins. IR -subunit, myelin basic protein, and microtubule-associated protein-2 are good substrates for IRS kinase. The kinase prefers Mn²⁺ (K_a=1.3 mM) as a metal cofactor. Mg²⁺ (K_a=3.3 mM) is only 30% as effective as Mn²⁺. The kinase activity is stimulated by basic polypeptides, with greater than 30-fold activation achieved with polylysine and protamine. Our results suggest that both serine/threonine and tyrosine phosphorylation are required for activation of IRS kinase. Serine phosphorylation is catalyzed by one of the casein kinases, whereas tyrosine phosphorylation is catalyzed by a membrane tyrosine kinase, possibly IR tyrosine kinase. (Mol Cell Biochem121: 167–174, 1993)     

Activation of a manganese-dependent membrane protein kinase by serine and tyrosine phosphorylation.

A Mn2(+)-dependent serine/threonine protein kinase from rat liver membranes copurifies with the insulin receptor (IR) on wheat germ agglutinin (WGA)-sepharose. The kinase is present in a nonactivated form in membranes but can be activated 20-fold by phosphorylating the WGA-sepharose fraction with casein kinase-1 (CK-1), casein kinase-2 (CK-2), or casein kinase-3 (CK-3). The activated kinase can use IR beta-subunit, myelin basic protein, and histones as substrates. Activation of the kinase seems to proceed by two or more steps. Sodium vanadate and Mn2+ are required in reaction mixtures for activation to be observed, whereas the tyrosine kinase-specific substrate, poly (glu, tyr), completely inhibits activation. These observations suggest that, in addition to serine/threonine phosphorylation by one of the casein kinases, activation of the Mn2(+)-dependent protein kinase also requires tyrosine phosphorylation. Such phosphorylation may be catalyzed by the IR tyrosine kinase.     

Phosphorylation of the high-mobility-group-like protein P1 by casein kinase-2

The nuclear protein P1 (molecular mass 53 kDa), found in all mammalian cell types and tissues so far tested, is an excellent substrate for casein kinase-2. The number of phosphate groups on P1 is 20-30/molecule; the phosphorylation sites are distributed throughout the molecule. The phosphate is present as serine phosphate and possibly threonine phosphate. Proteolytic digestion with Staphylococcus aureus V8 protease of 32P-labelled P1 both in vivo and in vitro revealed that casein kinase-2 may be one of the kinases responsible for the phosphorylation in vivo.     

Comparison of the phosphorylation of microtubule-associated protein tau by non-proline dependent protein kinases

Microtubule-associated protein tau from Alzheimer brain has been shown to be phosphorylated at several ser/thr-pro and ser/thr-X sites (Hasegawa, M. et al., J. Biol. Chem, 267, 17047–17054, 1992). Several proline-dependent protein kinases (PDPKs) (MAP kinase, cdc2 kinase, glycogen synthase kinase-3, tubulin-activated protein kinase, and 40 kDa neurofilament kinase) are implicated in the phosphorylation of the ser-thr-pro sites. The identity of the kinase(s) that phosphorylate that ser/thr-X sites are unknown. To identify the latter kinase(s) we have compared the phosphorylation of bovine tau by several brain protein kinases. Stoichiometric phosphorylation of tau was achieved by casein kinase-1, calmodulin-dependent protein kinase II, Gr kinase, protein kinase C and cyclic AMP-dependent protein kinase, but not with casein kinase-2 or phosphorylase kinase. Casein kinase-1 and calmodulin-dependent protein kinase II were the best tau kinases, with greater than 4 mol and 3 mol³²P incorporated, respectively, into each mol of tau. With the sequential addition of these two kinases,³²P incorporation approached 6 mol. Peptide mapping revealed that the different kinases largely phosphorylate different sites on tau. After phosphorylation by casein kinase-1, calmodulin-dependent protein kinase II, Gr kinase, cyclic AMP-dependent protein kinase and casein kinase-2, the mobility of tau isoforms as detected by SDS-PAGE was decreased. Protein kinase C phosphorylation did not produce such a mobility shift. Our results suggest that one or more of the kinases studied here may participate in the hyperphosphorylation of tau in Alzheimer disease. Such phosphorylation may serve to modulate the activaties of other tau kinases such as the PDPKs.Abbreviations PHF paired helical filaments - A-kinase cyclic AMP-dependent protein kinase - CaM kinase II calcium/calmodulin-dependent protein kinase II - C-kinase calcium-phospholipid-dependent protein kinase - CK-1 casein kinase-1 - CK-2 casein kinase-2 - Gr kinase calcium/calmodulin-dependent protein kinase from rat cerebellum - GSK-3 glycogen synthase kinase-3 - MAP kinase mitogen-activated protein kinase - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Phosphorylation and activation of p40 tyrosine kinase by casein kinase-1

Because examination of regulatory trans-phosphorylations can help elucidate the cellular functions of tyrosyl protein kinases, we have investigated the effects of phosphorylation by casein kinase-1 on the activity of the p40 tyrosyl protein kinase. We find that casein kinase-1 can phosphorylate the p40 tyrosyl kinase on serine and threonine residues, in part on a unique tryptic peptide. The phosphorylation induces a substantial increase in the tyrosyl protein kinase activity of p40, in contrast to most instances in which serine/threonine phosphorylation inhibits activity of tyrosyl protein kinases. These findings raise the possibility that p40 might be part of a protein phosphorylation network in which casein kinase-1 participates.     

Selectivity of 4,5,6,7-tetrabromobenzotriazole, an ATP site-directed inhibitor of protein kinase CK2 ('casein kinase-2')

The specificity of 4,5,6,7-tetrabromo-2-azabenzimidazole (TBB), an ATP/GTP competitive inhibitor of protein kinase casein kinase-2 (CK2), has been examined against a panel of 33 protein kinases, either Ser/Thr- or Tyr-specific. In the presence of 10 microM TBB (and 100 microM ATP) only CK2 was drastically inhibited (>85%) whereas three kinases (phosphorylase kinase, glycogen synthase kinase 3 beta and cyclin-dependent kinase 2/cyclin A) underwent moderate inhibition, with IC(50) values one--two orders of magnitude higher than CK2 (IC(50)=0.9 microM). TBB also inhibits endogenous CK2 in cultured Jurkat cells. A CK2 mutant in which Val66 has been replaced by alanine is much less susceptible to inhibition by TBB as well as by another ATP competitive inhibitor, emodin. These data show that TBB is a quite selective inhibitor of CK2, that can be used in cell-based assays.     

Partially dephosphorylated phosphopeptide AcSer(P)-Ser(P)-Ser(P) is an excellent substrate for casein kinase-2

The synthetic phosphopeptide AcSer(P)-Ser(P)-Ser(P), reproducing a recurrent feature of casein and other phosphoproteins, once partially dephosphorylated by acid phosphatase, serves as an efficient substrate for casein kinase-2. Previous dephosphorylation beyond 30% hinders subsequent phosphorylation and the entirely dephosphorylated peptide is not a substrate at all. The kinetic constants of the partially dephosphorylated phosphopeptide are much more favourable than those of the synthetic peptides SEEEAA, SSEE and SEE, the latter one being totally inert. Optimal phosphorylation occurs at pH values that ensure complete ionization of the phosphoseryl side chains. These data provide incontrovertible demonstration that phosphoserine can replace carboxylic amino acids as specificity determinant for CK-2, being more effective than glutamic acid itself.     

Structure and properties of casein kinase-2 from Saccharomyces cerevisiae. A comparison with the liver enzyme

A type-2 casein kinase (YCK-2), lacking the 25-kDa autophosphorylatable beta subunit characteristic of animal casein kinases-2, has been obtained in a nearly pure form from Saccharomyces cerevisiae and was compared with liver casein kinase-2 (LCK-2). A 22-kDa phosphorylatable protein, copurifying with YCK-2, can be removed by ultracentrifugation at low ionic strength and is shown by several criteria to be unrelated to the beta subunit of LCK-2. The native Mr of YCK-2, deprived of the 22-kDa phosphoprotein, is about 150 000. Limited proteolysis experiments show that YCK-2 included 37-kDa catalytic subunits, which can be converted into still active 35-kDa proteolytic derivatives. These data are consistent with a homotetrameric quaternary structure as opposed to the heterotetrameric subunit composition alpha 2 beta 2 of LCK-2 and other animal casein kinases-2. Although many properties of YCK-2 and LCK-2, including substrate specificity, inhibition by heparin, polyglutamic acid and quercetin and stimulation by polyamines, are similar; their stability under denaturing and dissociating conditions and their response to polybasic peptides are quite different. In particular YCK-2 is more readily denatured than LCK-2 by heating and exposure to urea, sodium dodecylsulphate and deoxycholate while its activity is inhibited by 100-150 mM NaCl, which conversely stimulates LCK-2 activity 2-3-fold. The Km value of the synthetic peptide substrate Ser-(Glu)5 for YCK-2 is not significantly changed by the addition of polylysine. On the contrary the Km value of the same peptide substrate for LCK-2 decreases approximately tenfold upon addition of polylysine, which also prevents the fast autophosphorylation of the kinase at its beta subunit. These data suggest that the beta subunit of animal CK-2 may play a role in determining both the stability of the enzyme and its regulation and that, consequently, the different properties of YCK-2 may be at least in part accounted for by its lack of beta subunits.     

Crystal structure of casein kinase-1, a phosphate-directed protein kinase.

The structure of a truncated variant of casein kinase-1 from Schizosaccharomyces pombe, has been determined in complex with MgATP at 2.0 A resolution. The model resembles the 'closed', ATP-bound conformations of the cyclin-dependent kinase 2 and the cAMP-dependent protein kinase, with clear differences in the structure of surface loops that impart unique features to casein kinase-1. The structure is of unphosphorylated, active conformation of casein kinase-1 and the peptide-binding site is fully accessible to substrate.