Geldanamycin-induced Lyn dissociation from aberrant Hsp90-stabilized cytosolic complex is an early event in apoptotic mechanisms in B-chronic lymphocytic leukemia.

Lyn, a tyrosine kinase belonging to the Src family, plays a key role as a switch molecule that couples the B-cell receptor to downstream signaling. In B-CLL cells, Lyn is overexpressed, anomalously present in the cytosol, and displays a high constitutive activity, compared with normal B lymphocytes. The aim of this work was to gain insights into the molecular mechanisms underlying these aberrant properties of Lyn, which have already been demonstrated to be related to defective apoptosis in B-cell chronic lymphocytic leukemia (B-CLL) cells. Herein, Lyn is described to be in an active conformation as integral component of an aberrant cytosolic 600-kDa multiprotein complex in B-CLL cells, associated with several proteins, such as Hsp90 through its catalytic domain, and HS1 and SHP-1L through its SH3 domain. In particular, Hsp90 appears tightly bound to cytosolic Lyn (CL), thus stabilizing the aberrant complex and converting individual transient interactions into stable ones. We also demonstrate that treatment of B-CLL cells with geldanamycin, an Hsp90 inhibitor already reported to induce cell death, is capable of dissociating the CL complex in the early phases of apoptosis and thus inactivating CL itself. These data identify the CL complex as a potential target for therapy in B-CLL.

Lyn-mediated mitochondrial tyrosine phosphorylation is required to preserve mitochondrial integrity in early liver regeneration.

Functional alterations in mitochondria such as overproduction of ROS (reactive oxygen species) and overloading of calcium, with subsequent change in the membrane potential, are traditionally regarded as pro-apoptotic conditions. Although such events occur in the early phases of LR (liver regeneration) after two-thirds PH (partial hepatectomy), hepatocytes do not undergo apoptosis but continue to proliferate until the mass of the liver is restored. The aim of the present study was to establish whether tyrosine phosphorylation, an emerging mechanism of regulation of mitochondrial function, participates in the response to liver injury following PH and is involved in contrasting mitochondrial pro-apoptotic signalling. Mitochondrial tyrosine phosphorylation, negligible in the quiescent liver, was detected in the early phases of LR with a trend similar to the events heralding mitochondrial apoptosis and was attributed to the tyrosine kinase Lyn, a member of the Src family. Lyn was shown to accumulate in an active form in the mitochondrial intermembrane space, where it was found to be associated with a multiprotein complex. Our results highlight a role for tyrosine phosphorylation in accompanying, and ultimately counteracting, mitochondrial events otherwise leading to apoptosis, hence conveying information required to preserve the mitochondrial integrity during LR.

Characterization of immune complexes of idiotypic catalytic and anti-idiotypic inhibitory antibodies in plasma of type 1 diabetic subjects.

An increase in proteolytic activity is an early common feature of diabetes, and is associated with the development of vascular complications. We performed an extensive proteomic investigation on plasma of type 1 diabetic subjects to discover why some of them apparently lacked any measurable proteolytic activity. Activity was found enclosed in immune complexes in which Fab/(Fab)(2) displayed a serine-like catalytic activity. Disaggregation of complexes by means of Protein G affinity chromatography led to the separation of free subunits of Fab, showing a specific amidolytic activity, from Fab that displayed activity on casein and remained closely complexed with whole IgG. On both types of Fab the serine catalytic site appeared to be the same, being located in close vicinity to the antigen-binding site. The distinct substrate specificity was due to the different conformation adopted by the catalytic site depending on the structure of Fab/(Fab)(2), whether in complexes or as free subunits. Catalytic Fab/(Fab)(2) originated from idiotypic antibodies developed against Grp94, identified as the primary antigen covalently complexed with Fab. Whole IgG present in immune complexes were instead mostly formed with anti-idiotypic antibodies developed against the adduct of Fab/(Fab)(2) with Grp94, and were responsible for blocking any catalytic activity. In dot-blot experiments with native Grp94, we confirmed that in any diabetic plasma circulated anti-Grp94, idiotypic, and anti-idiotypic antibodies.

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 Golgi-associated 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.

Recognition of lysine-rich peptide ligands by murine cortactin SH3 domain: CD, ITC, and NMR studies.

Cortactin is a ubiquitous actin-binding protein that regulates various aspects of cell dynamics and is implicated in the pathogenesis of human neoplasia. The sequence of cortactin contains a number of signaling motifs and an SH3 domain at the C-terminus, which mediates the interaction of the protein with several partners, including Shank2. A recombinant protein, comprising the murine cortactin SH3 domain fused to GST (GST-SH3(m-cort)), was prepared and used to assess the domain-binding affinity of potential peptide-ligands reproducing the proline-rich regions of human HPK1 and Shank2 proteins. The key residues involved in the SH3(m-cort) domain recognition were identified by three different approaches: non-immobilized ligand interaction assay by circular dichroism, isothermal titration calorimetry, and nuclear magnetic resonance. Our results show that the classical PxxPxK class II binding motif is not sufficient to mediate the interaction with GST-SH3(m-cort), an event that depends on the presence of additional basic residues located at either the N- or the C-terminus of the PxxPxK motif. Especially effective in promoting the peptide binding is a Lys residue at the -5 position, a determinant present in both P2 (HPK1 394-403) and S1 (Shank2 1168-1189) peptides. GST-SH3(m-cort) exhibits the highest affinity toward peptide S1, which contains additional Lys residues at the -3, -5, and -7 positions, indicating that the optimal consensus motif may be KPPxPxKxKxK. These results are supported by the in silico models of SH3(m-cort) complexed with P2 or S1, which highlight the domain residues that interact with the recognition determinants of the peptide-ligand and cooperate in binding stabilization.

Analysis of tyrosine-phosphorylated proteins in rat brain mitochondria.

Mitochondrial protein phosphorylation is emerging as a central event in mitochondrial signaling. In particular, tyrosine phosphorylation is proving to be an unappreciated mechanism involved in regulation of mitochondrial functions. Tyrosine kinases and phosphatases have been identified in mitochondrial compartments and there is a steadily increasing number of new identified tyrosine-phosphorylated proteins implicated in a wide spectrum of mitochondrial functions. The deciphering of the tyrosine phosphorylation signaling in mitochondria is strictly linked to the definition of the entire mitochondrial tyrosine phosphoproteome. This chapter describes methods to analyze tyrosine phosphorylation in brain mitochondria: identification of new substrates by biochemical and mass spectrometry approaches and bioinformatic tools to analyze the potential effect of tyrosine phosphorylation on the structure/activity of a protein.