Luca Cesaro

Chronic lymphocytic leukemia B cells contain anomalous Lyn tyrosine kinase, a putative contribution to defective apoptosis

B cell chronic lymphocytic leukemia (B-CLL) is a neoplastic disorder characterized by accumulation of B lymphocytes due to uncontrolled growth and resistance to apoptosis. Analysis of B cells freshly isolated from 40 patients with chronic lymphocytic leukemia demonstrated that the Src kinase Lyn, the switch molecule that couples the B cell receptor to downstream signaling, displays anomalous properties. Lyn is remarkably overexpressed at the protein level in leukemic cells as compared with normal B lymphocytes, with a substantial aliquot of the kinase anomalously present in the cytosol. Whereas in normal B lymphocytes Lyn activation is dependent on B cell-receptor stimulation, in resting malignant cells, the constitutive activity of the kinase accounts for high basal protein tyrosine phosphorylation and low responsiveness to IgM ligation. Addition of the Lyn inhibitors PP2 and SU6656 to leukemic cell cultures restores cell apoptosis, and treatment of malignant cells with drugs that induce cell apoptosis decreases both activity and amount of the tyrosine kinase. These findings suggest a direct correlation between high basal Lyn activity and defects in the induction of apoptosis in leukemic cells. They also support a critical role for Lyn in B-CLL pathogenesis and identify this tyrosine kinase as a potential therapeutic target.

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 noncanonical sequence phosphorylated by casein kinase 1 in beta-catenin may play a role in casein kinase 1 targeting of important signaling proteins.

Protein kinase casein kinase 1 (CK1) phosphorylates Ser-45 of β-catenin, “priming” the subsequent phosphorylation by glycogen synthase-3 of residues 41, 37, and 33. This concerted phosphorylation of β-catenin signals its degradation and prevents its function in triggering cell division. The sequence around Ser-45 does not conform to the canonical consensus for CK1 substrates, which prescribes either phosphoamino acids or acidic residues in position n-3 from the target serine. However, the β-catenin sequence downstream from Ser-45 is very similar to a sequence recognized by CK1 in nuclear factor for activated T cells 4. The common features include an SLS motif followed two to five residues downstream by a cluster of acidic residues. Synthetic peptides reproducing residues 38-65 of β-catenin were assayed with purified rat liver CK1 or recombinant CK1α and CK1αL from zebrafish. The results demonstrate that SLS and acidic cluster motifs are crucial for CK1 recognition. Pro-44 and Pro-52 are also important for efficient phosphorylation. Similar results were obtained with the different isoforms of CK1. Phosphorylation of mutants of full-length recombinant β-catenin from zebrafish confirmed the importance of the SLS and acidic cluster motifs. A search for proteins with similar motifs yielded, among other proteins, adenomatous polyposis coli, previously found to be phosphorylated by CK1. There is a strong correlation of β-catenin mutations found in thyroid tumors with the motifs recognized by CK1 in this protein.

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 (Tyr184) 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 Tyr184(DEDADIY184DEEDYDL) is phosphorylated by CK2, albeit with much higher K m (384 versus 4.3 μm) and lower V max (8.4versus 1,132 nmol·min−1·mg−1) than its derivative with Tyr184 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.

Identification of new tyrosine phosphorylated proteins in rat brain mitochondria.

Utilizing immunoaffinity enrichment of phosphotyrosine‐containing peptides coupled to mass spectrometric analysis we detected new tyrosine phosphorylated proteins in rat brain mitochondria after peroxovanadate treatment. By bioinformatic predictions we provide suggestions about the potential role of tyrosine phosphorylation in mitochondrial physiology. Our results indicate a primary role of tyrosine phosphorylation in regulating energy production at the mitochondrial level. Moreover, tyrosine phosphorylation might regulate the mitochondrial membrane permeability targeting protein complexes containing ADP/ATP translocase, VDAC, creatine kinase and hexokinase.

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.

Structure-function analysis of yeast piD261/Bud32, an atypical protein kinase essential for normal cell life

The Saccharomyces cerevisiae YGR262c/BUD32 gene, whose disruption causes a severe pleiotropic phenotype, encodes a 261-residue putative protein kinase, piD261, whose structural homologues have been identified in a variety of organisms, including humans, and whose function is unknown. We have demonstrated previously that piD261, expressed in Escherichia coli as a recombinant protein, is a Ser/Thr kinase, as judged by its ability to autophosphorylate and to phosphorylate casein. Here we describe a mutational analysis showing that, despite low sequence similarity, the invariant residues representing the signature of protein kinases are conserved in piD261 and in its structural homologues, but are embedded in an altered context, suggestive of unique mechanistic properties. Especially noteworthy are: (i) three unique inserts of unknown function within the N-terminal lobe, (ii) the lack of a lysyl residue which in all other Ser/Thr kinases participates in the catalytic event by interacting with the transferred ATP gamma-phosphate, and which in piD261 is replaced by a threonine, and (iii) an exceedingly short activation loop including two serines, Ser-187 and Ser-189, whose autophosphorylation accounts for the appearance of an upshifted band upon SDS/PAGE. A mutant in which these serines are replaced by alanines was devoid of the upshifted band and displayed reduced catalytic activity. This would include piD261 in the category of protein kinases activated by phosphorylation, although it lacks the RD (Arg-Asp) motif which is typical of these enzymes.

Motif Analysis of Phosphosites Discloses a Potential Prominent Role of the Golgi Casein Kinase (GCK) in the Generation of Human Plasma Phospho-Proteome

By comparing the recurrent features of sequences surrounding 86 Ser/Thr residues phosphorylated in peptides from human plasma collected from literature with those generated from the whole human phosphoproteome, and from repertoires of validated substrates of the acidophilic protein kinases CK2 and Golgi casein kinase (GCK), the following conclusions can be drawn: (i) the contribution of Pro-directed and basophilic kinases to the plasma phosphoproteome is negligible, if any, while the contribution of acidophilic kinases is by far predominant; (ii) the plasma weblogo profile is closely reminiscent of that generated by GCK in its substrates, while it neatly differentiates from that generated by CK2; (iii) 58 plasma phosphosites out of 86 display the canonical consensus for GCK (S/T-x-E/pS), while that for CK2 (S/T-x-x-E/D/pS) is found in 15 peptides, all of which also conform to the GCK signature. These observations, in conjunction with a very similar situation disclosed by analyzing the phosphopeptides of the human cerebrospinal fluid collected from literature, support the view that GCK may play a major role in the phosphorylation of proteins secreted into body fluids.

Functional Complementation in Yeast Allows Molecular Characterization of Missense Argininosuccinate Lyase Mutations

Deficiency of argininosuccinate lyase (ASL) causes argininosuccinic aciduria, an urea cycle defect that may present with a severe neonatal onset form or with a late onset phenotype. To date phenotype-genotype correlations are still not clear because biochemical assays of ASL activity correlate poorly with clinical severity in patients. We employed a yeast-based functional complementation assay to assess the pathogenicity of 12 missense ASL mutations, to establish genotype-phenotype correlations, and to screen for intragenic complementation. Rather than determining ASL enzyme activity directly, we have measured the growth rate in arginine-free medium of a yeast ASLnull strain transformed with individual mutant ASL alleles. Individual haploid strains were also mated to obtain diploid, “compound heterozygous” yeast. We show that the late onset phenotypes arise in patients because they harbor individual alleles retaining high residual enzymatic activity or because of intragenic complementation among different mutated alleles. In these cases complementation occurs because in the hybrid tetrameric enzyme at least one active site without mutations can be formed or because the differently mutated alleles can stabilize each other, resulting in partial recovery of enzymatic activity. Functional complementation in yeast is simple and reproducible and allows the analysis of large numbers of mutant alleles. Moreover, it can be easily adapted for the analysis of mutations in other genes involved in urea cycle disorders.

Spleen protein tyrosine kinases TPK-IIB and CSK display different immunoreactivity and opposite specificities toward c-src-derived peptides

Polyclonal antibodies have been raised against two synthetic peptides reproducing the 48–64 and 353–369 sequences of CSK, a protein tyrosine kinase implicated in the down‐regulation of src‐related protein kinases. Both antibodies specifically recognize recombinant CSK and a CSK‐related 49 kDa protein tyrosine kinase present in spleen but they do not cross‐react with purified TPK‐IIB, a spleen protein tyrosine kinase sharing with CSK catalytic activity toward src kinases and incapability to autophosphorylate. CSK and TPK‐IIB once resolved from each other by heparin‐Sepharose affinity chromatography, display opposite specificities toward synthetic peptides reproducing the sequences around the main phosphoacceptor residues of pp6Ocsrc , namely Tyr‐416 and Tyr‐527. These data support the view that TPK‐IIB and CSK may exert opposite effects on the activity of src‐related protein tyrosine kinases.