A "SYDE" effect of hierarchical phosphorylation: possible relevance to the cystic fibrosis basic defect.

The motif "SYDE", incorporating the protein kinase CK2 consensus sequence (S-x-x-E) has been found to be phosphorylated at both its serine and tyrosine residues in several proteins. Of special interest is the case of cystic fibrosis Transmembrane-conductance Regulator (CFTR), where this motif is close to the residue (F508), whose deletion is the by far commonest cause of cystic fibrosis. Intriguingly, however, CFTR S511 cannot be phosphorylated by CK2 to any appreciable extent. Using a number of peptide substrates encompassing the CFTR "SYDE" site we have recently shown that: (1) failure of CK2 to phosphorylate the S(511)YDE motif is due to the presence of Y512; (2) CK2 readily phosphorylates S511 if Y512 is replaced by a phospho-tyrosine; (3) the Src family protein tyrosine kinase Lyn phosphorylates Y512 in a manner that is enhanced by the deletion of F508. These data, in conjunction with the recent observation that by inhibiting CK2 the degradation of F508delCFTR is reduced, lead us to hypothesize that the hierarchical phosphorylation of the motif SYDE by the concerted action of protein tyrosine kinases and CK2 is one of the mechanisms that cooperate to the premature degradation of F508delCFTR.

Protein kinase CK2: a newcomer in the 'druggable kinome'.

The acronym CK2 (derived from the misnomer 'casein kinase' 2) denotes one of the most pleiotropic members of the eukaryotic protein kinase superfamily, characterized by an acidic consensus sequence in which a carboxylic acid (or pre-phosphorylated) side chain at position n+3 relative to the target serine/threonine residue plays a crucial role. The latest repertoire of CK2 substrates includes approx. 300 proteins, but the analysis of available phosphopeptide databases from different sources suggests that CK2 alone may be responsible for the generation of a much larger proportion (10-20%) of the eukaryotic phosphoproteome. Although for the time being CK2 is not included among protein kinases whose inhibitors are in clinical practice or in advanced clinical trials, evidence is accumulating that elevated CK2 constitutive activity co-operates to induce a number of pathological conditions, including cancer, infectious diseases, neurodegeneration and cardiovascular pathologies. The development and usage of cell-permeant, selective inhibitors discloses a scenario whereby CK2 plays a global anti-apoptotic role, which under special circumstances may lead to untimely and pathogenic cell survival.

Autocatalytic tyrosine-phosphorylation of protein kinase CK2 alpha and alpha' subunits: implication of Tyr182.

CK2 is a pleiotropic and constitutively active serine/threonine protein kinase composed of two catalytic (alpha and/or alpha') and two regulatory beta-subunits, whose mechanism of modulation is still obscure. Here we show that CK2 alpha/alpha' subunits undergo intermolecular (trans) tyrosine-autophosphorylation, which is dependent on intrinsic catalytic activity and is suppressed by the individual mutation of Tyr182, a crucial residue of the activation loop, to phenylalanine. At variance with serine-autophosphorylation, tyrosine-autophosphorylation of CK2alpha is reversed by ADP and GDP and is counteracted by the beta-subunit and by a peptide reproducing the activation loop of CK2alpha/alpha' (amino acids 175-201). These results disclose new perspectives about the mode of regulation of CK2 catalytic subunits.

Distribution of protein disulphide isomerase in rat liver mitochondria.

Here we report the localization of protein disulphide isomerase (PDI) in the mitochondrial compartments, comparing it with that of thioredoxin reductase. The latter enzyme is present mostly in the matrix, whereas PDI is located at the level of the outer membrane. We characterize the different submitochondrial fractions with specific marker enzymes. PDI, whether isolated from whole mitochondria or from purified outer membranes, exhibits the same electrophoretic mobility, indicating identical molecular masses. Moreover, immunoblot analysis with monoclonal anti-PDI antibody shows immunoreactivity only with the microsomal PDI, indicating the specificity of the mitochondrial isoform. The significance of these findings is discussed with reference to the potential role of PDI and thioredoxin reductase in regulating the mitochondrial functions dependent on the thiol-disulphide transition.

Generation of mutants of CK2alpha which are dependent on the beta-subunit for catalytic activity.

To shed light on the structural features underlying high constitutive activity of protein kinase CK2 a number of mutants of the human CK2alpha-subunit altered in the interactions between the N-terminal segment and the activation loop have been generated and shown to be defective in catalytic activity. In particular the truncated mutant delta2-12 displays under standard conditions an almost complete loss of catalytic activity accounted for by a dramatic rise in its Km forATP (from 10 to 206 microM) and a reduced Kcat. Such a drop in efficiency is paralleled by conformational disorganization, as judged from Superdex 75 gel filtration profile. Both catalytic properties and gel filtration behaviour similar to those of wild type CK2alpha were restored upon association with the regulatory beta-subunit, suggesting that constitutive activity is conferred to CK2alpha and to CK2 holoenzyme through different molecular mechanisms. In the holoenzyme an assumable release of tension at the backbone of Ala-193 (as seems to be indicated by a comparison of the crystal structures of maize CK2alpha alone vs. a CK2alpha-beta peptide complex) may result in the ability of the activation loop to adopt its proper conformation independently of interactions with the N-terminal segment.

Isolation, purification, and characterization of a rat liver mitochondrial protein disulfide isomerase.

The isolation and purification to electrophoretical homogeneity and characterization of a protein disulfide isomerase from rat liver mitochondria is reported. The purified enzyme exhibits a single band on sodium dodecylsulfatepolyacrylamide gel electrophoresis with an apparent molecular weight of approximately 54 kDa. Comparatively, the microsomal form shows an apparent molecular weight of 57 kDa indicating that the two forms are slightly different. The antibody raised against the microsomal isoform does not recognize the mitochondrial enzyme. To characterize the enzyme, different classical methodologies utilized for protein disulfide isomerase estimation have been adopted. The isolated enzyme is active with all of them, indicating that it comprises all the features of a typical protein disulfide isomerase. At the mitochondrial level the enzyme appears mostly localized at the membrane level. Its potential involvement in mitochondrial membrane permeability control is also discussed.

Tyrosine versus serine/threonine phosphorylation by protein kinase casein kinase-2. A study with peptide substrates derived from immunophilin Fpr3.

Protein kinase casein kinase-2 (CK2) is a spontaneously active, ubiquitous, and pleiotropic enzyme that phosphorylates seryl/threonyl residues specified by multiple negatively charged side chains, the one at position n + 3 being of crucial importance (minimum consensus S/T-x-x-E/D/S(P)/T(P). Recently CK2 has been reported to catalyze phosphorylation of the yeast nucleolar immunophilin Fpr3 at a tyrosyl residue (Tyr(184)) fulfilling the consensus sequence of Ser/Thr substrates (Wilson, L.K., Dhillon, N., Thorner, J., and Martin, G.S. (1997) J. Biol. Chem. 272, 12961-12967). Here we show that, by contrast to other tyrosyl peptides fulfilling the consensus sequence for CK2, a peptide reproducing the sequence around Fpr3 Tyr(184) (DEDADIY(184)DEEDYDL) is phosphorylated by CK2, albeit with much higher K(m) (384 versus 4. 3 microM) and lower V(max) (8.4 versus 1,132 nmol.min(-1).mg(-1)) than its derivative with Tyr(184) replaced by serine. The replacement of Asp at position n + 1 with alanine and, to a lesser extent, of Ile at n - 1 with Asp are especially detrimental to tyrosine phosphorylation as compared with serine phosphorylation, which is actually stimulated by the Ile to Asp modification. In contrast the replacement of Glu at n + 3 with alanine almost suppresses serine phosphorylation but not tyrosine phosphorylation. It can be concluded that CK2 is capable to phosphorylate, under special circumstances, tyrosyl residues, which are specified by structural features partially different from those that optimize Ser/Thr phosphorylation.

CK2: a protein kinase in need of control.

Protein kinase CK2 is a heterotetrameric alpha2beta2 Ser/Thr protein kinase with some features unusual among the eukaryotic protein kinases: (1) CK2 recognizes phosphoacceptor sites specified by several acidic determinants; (2) CK2 can use both ATP and GTP as phosphoryl donors; and (3) the regulatory properties of CK2 are poorly understood; it is insensitive to any known second messenger and displays high basal activity. To gain insight into CK2 regulation and to understand its unusual properties, site-directed mutagenesis experiments on both subunits and X-ray crystallographic studies of the catalytic alpha-subunit were performed. The noncatalytic beta-subunit has at least three functions: (1) it protects the alpha-subunit against denaturing agents or conditions; (2) it alters the substrate specificity of the alpha-subunit; and (3) it modulates the activity of the enzyme, i.e., depending on the substrate, it increases or decreases the activity of the alpha-subunit. Mutagenesis experiments revealed that an acidic stretch between amino acids 55 and 64 has a down-regulatory and autoinhibitory function. Mutational analysis of the alpha-subunit has revealed a network of unique basic residues that are responsible for the recognition of phosphoacceptor substrates and for down-regulation by the beta-subunit and by polyanionic inhibitors. The resolution of the crystal structure of Zea mays CK2 alpha-subunit has disclosed the structural features that are responsible for high basal activity and for unusual response to nucleotide analogs. The increasing knowledge of CK2 structure-function relationships will allow the design of highly selective inhibitors of this pleiotropic kinase with oncogenic potential.

A multifunctional network of basic residues confers unique properties to protein kinase CK2.

Protein kinase CK2 is characterized by a number of features, including substrate specificity, inhibition by polyanionic compounds and intrasteric down-regulation by its beta-subunit, which denote a special aptitude to interact with negatively charged ligands. This situation may reflect the presence in CK2 catalytic subunits of several basic residues that are not conserved in the majority of other protein kinases. Some of these residues, notably K49 in the 'Gly rich loop', K74, K75, K76, K77, K79, R80, K83 in the 'Lys rich segment' and R191, R195, K198 in the 'p+1 loop', have been shown by mutational studies to be implicated to various extents and with distinct roles in substrate recognition, inhibition by heparin and by pseudosubstrate and instrasteric regulation. Molecular modelization based on crystallographic data provide a rationale for the biochemical observations, showing that several of these basic residues are clustered around the active site where they make contact with individual acidic residues of the peptide substrate. They can also mediate the effect of polyanionic inhibitors (e.g. heparin) and of regulatory elements present in the beta-subunit, in the N terminal segment of the catalytic subunit and possibly in other proteins interacting with CK2. Our data also disclose a unique mode of binding of the phosphoacceptor substrate which bridges across the catalytic cleft making contacts with both the lower and upper lobes of CK2.

Linear and cyclic peptides as substrates for Lyn tyrosine kinase.

Two Tyr residues are supposed to play a crucial role in the regulation of protein tyrosine kinases of the Src family. Autophosphorylation of Src Tyr416 correlates with enzyme activation, while phosphorylation of C-terminal Tyr527 by Csk gives rise to inactive forms of Src kinases. It has previously been demonstrated that the Src-like tyrosine kinase expressed by the oncogene lyn displays a particularly high affinity (Km 20 microm) toward the dimeric linear and cyclic derivatives of the heptapeptide H-Glu-Asp-Asn-Glu-Tyr-Thr-Ala-OH which reproduces the main autophosphorylation site of most of the Src enzymes. Under the experimental conditions used only one Tyr residue of the dimeric sequence can be phosphorylated [P. Ruzza, A. Calderan, B.Filippi, B. Biondi, A. Donella Deana, L. Cesaro, L. A. Pinna & G. Borin (1995) Int. J. Peptide Protein Res. 45, 529-539]. The present study addresses the problem of the efficiency displayed by Lyn towards the two Tyr residues located at positions 5 and 12 of the dimeric peptide. To this purpose, two tetradecapeptides were synthesized by the classical solution method, each containing one of the two Tyr residues alternatively replaced by Phe, and the corresponding univocal cyclic form. A possible correlation between the different structural properties induced by the modifications of the native sequence and the ability of the peptides to act as Lyn substrates was noted. The kinetic data obtained indicate that Lyn phosphorylates the residues located at different positions in the two linear analogues differently. In particular, while the Tyr5, Phe12 derivative presents a Km value similar to those obtained for the dimeric linear and cyclic unmodified analogues, the Km value of the Phe5, Tyr12 derivative is two-fold higher than those found for the above-mentioned peptides. Moreover, as previously reported for the linear and cyclic dimeric forms of the native sequence, in the mono-tyrosine containing series of dimers the still conformationally flexible cyclic derivative shows a phosphorylation efficiency two-fold higher than those found for the linear derivatives.