Anna Maria Brunati

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.

Sequential phosphorylation of protein band 3 by Syk and Lyn tyrosine kinases in intact human erythrocytes: identification of primary and secondary phosphorylation sites

Treatment of intact human erythrocytes with pervanadate induces Tyr (Y)-phosphorylation of the transmembrane protein band 3; in parallel, the activity of the immunoprecipitated tyrosine kinases Syk and Lyn is increased. When erythrocytes are incubated with pervanadate together with PP1, a specific inhibitor of Src kinases, including Lyn, the Y-phosphorylation of band 3 is only partially reduced. Indeed, the PP1-resistant phosphorylation of band 3 precedes and is a prerequisite for its coimmunoprecipitation with Lyn, which interacts with the phosphoprotein via the SH2 domain of the enzyme, as proven by binding competition experiments. Upon recruitment to primarily phosphorylated band 3, Lyn catalyzes the secondary phosphorylation of the transmembrane protein. These data are consistent with the view that band 3 is phosphorylated in intact erythrocytes by both PP1-resistant (most likely Syk) and PP1-inhibited (most likely Lyn) tyrosine kinases according to a sequential phosphorylation process. Similar radiolabeled peptide maps are obtained by tryptic digestion of (32)P-band 3 isolated from either pervanadate-treated erythrocytes or red cell membranes incubated with exogenous Syk and Lyn. It has also been demonstrated by means of mass spectrometry that the primary phosphorylation of band 3 occurs at Y8 and Y21, while the secondary phosphorylation affects Y359 and Y904. (Blood. 2000;96:1550-1557)

Catalase Takes Part in Rat Liver Mitochondria Oxidative Stress Defense

Highly purified rat liver mitochondria (RLM) when exposed to tert-butylhydroperoxide undergo matrix swelling, membrane potential collapse, and oxidation of glutathione and pyridine nucleotides, all events attributable to the induction of mitochondrial permeability transition. Instead, RLM, if treated with the same or higher amounts of H2O2 or tyramine, are insensitive or only partially sensitive, respectively, to mitochondrial permeability transition. In addition, the block of respiration by antimycin A added to RLM respiring in state 4 conditions, or the addition of H2O2, results in O2 generation, which is blocked by the catalase inhibitors aminotriazole or KCN. In this regard, H2O2 decomposition yields molecular oxygen in a 2:1 stoichiometry, consistent with a catalatic mechanism with a rate constant of 0.0346 s-1. The rate of H2O2 consumption is not influenced by respiratory substrates, succinate or glutamate-malate, nor by N-ethylmaleimide, suggesting that cytochrome c oxidase and the glutathione-glutathione peroxidase system are not significantly involved in this process. Instead, H2O2 consumption is considerably inhibited by KCN or aminotriazole, indicating activity by a hemoprotein. All these observations are compatible with the presence of endogenous heme-containing catalase with an activity of 825 ± 15 units, which contributes to mitochondrial protection against endogenous or exogenous H2O2. Mitochondrial catalase in liver most probably represents regulatory control of bioenergetic metabolism, but it may also be proposed for new therapeutic strategies against liver diseases. The constitutive presence of catalase inside mitochondria is demonstrated by several methodological approaches as follows: biochemical fractionating, proteinase K sensitivity, and immunogold electron microscopy on isolated RLM and whole rat liver tissue.

Characterization and location of Src-dependent tyrosine phosphorylation in rat brain mitochondria

Analysis of protein phosphorylation in highly purified rat brain mitochondria revealed the presence of several alkali-stable phosphoproteins whose phosphorylation markedly increases upon treatment with peroxovanadate and Mn(2+), a property indicating tyrosine phosphorylation. These include three prominent bands, with apparent sizes of 50, 60, and 75 kDa, which are detectable by anti-phosphotyrosine. Tyrosine phosphorylation disappears when mitochondria are treated with PP2, an inhibitor of the Src kinase family, suggesting the presence of members of this family in rat brain mitochondria. Immunoblotting and immunoprecipitation assays of mitochondrial lysates confirmed the presence of Fyn, Src and Lyn kinases, as well as Csk, a protein kinase which negatively controls the activity of the Src kinase family. Results show that tyrosine-phosphorylated proteins are membrane-bound and that they are located on the inner surface of the outer membrane and/or the external surface of the inner membrane. Instead, Src tyrosine kinases are mainly located in the intermembrane space - in particular, as revealed by immunogold experiments for Lyn kinase, in the cristal lumen. Rat brain mitochondria were also found to possess a marked level of tyrosine phosphatase activity, strongly inhibited by peroxovanadate.

Multiple phosphorylation of α-synuclein by protein tyrosine kinase Syk prevents eosin-induced aggregation

The presence of aggregated α‐synuclein molecules is a common denominator in a variety of neurodegenerative disorders. Here, we show that α‐synuclein (α‐syn) is an outstanding substrate for the protein tyrosine kinase p72syk(Syk), which phosphorylates three tyrosyl residues in its C‐terminal domain (Y‐125, Y‐133, and Y‐136), as revealed from experiments with mutants where these residues have been individually or multiply replaced by phenylalanine. In contrast, only Y‐125 is phosphorylated by Lyn and c‐Fgr. Eosin‐induced multimerization is observed with wildtype α‐syn, either phosphorylated or not by Lyn, and with all its Tyr to Phe mutants but not with the protein previously phosphorylated by Syk. Syk‐mediated phosphorylation also counteracts α‐syn assembly into filaments as judged from the disappearance of α‐syn precipitated upon centrifugation at 100,000 × g. We also show that Syk and α‐syn colocalize in the brain, and upon cotransfection in Chinese hamster ovary cells, α‐syn becomes Tyr‐phosphorylated by Syk. Moreover, Syk and α‐syn interact with each other as judged from the mammalian two‐hybrid system approach. These data suggest that Syk or tyrosine kinase(s) with similar specificity may play an antineurodegenerative role by phosphorylating α‐syn, thereby preventing its aggregation.

Polycation-dependent, Ca2+-antagonized phosphorylation of calmodulin by casein kinase-2 and a spleen tyrosine protein kinase

Ten distinct protein kinases have been tested for their ability to phosphorylate calmodulin. Only casein kinase‐2 and a spleen tyrosine protein kinase (TPK‐III) proved effective, their phosphorylation efficiency being dramatically enhanced by histones and other polybasic peptides while being depressed by 50 μM Ca2+. Phosphorylation by CK‐2 takes place with a Km of 12 μM calmodulin, leading to the incorporation of more than 1.5 mol P/mol substrate. Ser81 and Thr79 are among the residues affected. On the other hand, the two tyrosyl residues of calmodulin are both phosphorylated by TPK‐III, Tyr99 being preferred over Tyr138.

Ser-256 phosphorylation dynamics of Aquaporin 2 during maturation from the ER to the vesicular compartment in renal cells

Aquaporin 2 (AQP2) phosphorylation at Ser‐256 by protein kinase A (PKA) is a key signal for vasopressin‐stimulated AQP2 insertion into the plasma membrane in renal cells. This study underscores the possible role of phosphorylation at Ser‐256 in regulating AQP2 maturation. AQP2‐transfected renal CD8 cells were incubated with brefeldin A (BFA) to accumulate newly synthesized AQP2 in the endoplasmic reticulum (ER), and AQP2 flow from ER to the vesicular compartment was analyzed after BFA washout. We found that a) in the ER, AQP2 is weakly phosphorylated; b) the amount of phosphorylated AQP2 (p‐AQP2) at Ser‐256 increased significantly during transit in the Golgi, even in the presence of the PKA inhibitor H89; and c) AQP2 transport from the Golgi to the vasopressin‐regulated vesicular compartment occurred with a concomitant decrease in p‐AQP2 at Ser‐256. These results support the hypothesis that AQP2 transition in the Golgi apparatus is associated with a PKA‐independent increase in AQP2 phosphorylation at Ser‐256. Conversely, impaired constitutive phosphorylation in a Golgiassociated compartment occurring in cells expressing mutated S256A‐AQP2 or E258K‐AQP2 causes phosphorylation‐defective AQP2 routing to lysosomes. This result might explain the molecular basis of the dominant form of nephrogenic diabetes insipidus caused by the mutation E258K‐AQP2, in which the phenotype is caused by an impaired routing of AQP2.

Interaction of genistein with the mitochondrial electron transport chain results in opening of the membrane transition pore.

Genistein, a natural isoflavone present in soybeans, is a potent agent in the prophylaxis and treatment of cancer. Addition of genistein to isolated rat liver mitochondria (RLM) induces swelling, loss of membrane potential and release of accumulated Ca2+. These changes are Ca2+-dependent and are prevented by cyclosporin A (CsA) and bongkrekic acid (BKA), two classical inhibitors of the mitochondrial permeability transition (MPT). Induction of the MPT by genistein is accompanied by oxidation of thiol groups and pyridine nucleotides. The reducing agent dithioerythritol and the alkylating agent N-ethylmaleimide (NEM) completely prevent the opening of the transition pore, thereby emphasizing that the effect of the isoflavone correlates with the mitochondrial redox state. Further analyses showed that genistein induces the MPT by the generation of reactive oxygen species (ROS) due to its interaction with the respiratory chain at the level of mitochondrial complex III.

Tyrosine phosphatase activity in mitochondria: presence of Shp-2 phosphatase in mitochondria

Tyrosine phosphorylation by unidentified enzymes has been observed in mitochondria, with recent evidence indicating that non-receptorial tyrosine kinases belonging to the Src family, which represent key players in several transduction pathways, are constitutively present in mitochondria. The extent of protein phosphorylation reflects a coordination balance between the activities of specific kinases and phophatases. The present study demonstrates that purified rat brain mitochondria possess endogenous tyrosine phosphatase activity. Mitochondrial phosphatases were found to be capable of dephosphorylating different exogenous substrates, including paranitrophenylphosphate, 32P-poly(Glu-Tyr)4:1 and 32P-angiotensin. These activities are strongly inhibited by peroxovanadate, a well-known inhibitor of tyrosine phosphatases, but not by inhibitors of alkali or Ser/Thr phosphatases, and mainly take place in the intermembrane space and outer mitochondrial membrane. Using a combination of approaches, we identified the tyrosine phosphatase Shp-2 in mitochondria. Shp-2 plays a crucial role in a number of intracellular signalling cascades and is probably involved in several human diseases. It thus represents the first tyrosine phosphatase shown to be present in mitochondria.

Site specificity of p72syk protein tyrosine kinase: efficient phosphorylation of motifs recognized by Src homology 2 domains of the Src family.

Protein tyrosine kinase p72 syk purified from rat spleen has been assayed for its ability to phosphorylate a number of peptide substrates derived from naturally occurring phospho‐acceptor sites. The phosphorylation efficiency is extremely variable, depending on the peptide sequence, with K m values in the 3–1500 μM range. The by far best peptide substrates, with K m values of 3 and 4 μM are those reproducing the phospho‐acceptor sites of Vav and HS1 proteins, respectively. These sites include multiple acidic residues flanking tyrosine on both sides and they also display the consensus sequences (YEDL and YEEV) preferred by the SH2 domains of the Src family. Alteration of this consensus in the HS1 peptide, by replacing either the glutamic acid or valine, also reduces the phosphorylation efficiency by p72 syk . Also the replacement of acidic residues at position −1 and, to a lesser extent at positions −3 and −4 (but not at positions +3 and +5) are detrimental. These observations may suggest a role of p72 syk in the recruitment of ligands/substrates for the Src family enzymes. We also show that the HS1 peptide can be used for the specific monitoring of p72 syk since neither the two Src‐related c‐Fgr and Lyn kinases (needing a hydrophobic instead of acidic residue at position −1) nor CSK appreciably phosphorylate it.