Wnt5a induces ROR1 to complex with HS1 to enhance migration of chronic lymphocytic leukemia cells.

ROR1 (receptor tyrosine kinase-like orphan receptor 1) is a conserved, oncoembryonic surface antigen expressed in chronic lymphocytic leukemia (CLL). We found that ROR1 associates with hematopoietic-lineage-cell-specific protein 1 (HS1) in freshly isolated CLL cells or in CLL cells cultured with exogenous Wnt5a. Wnt5a also induced HS1 tyrosine phosphorylation, recruitment of ARHGEF1, activation of RhoA and enhanced chemokine-directed migration; such effects could be inhibited by cirmtuzumab, a humanized anti-ROR1 mAb. We generated truncated forms of ROR1 and found its extracellular cysteine-rich domain or kringle domain was necessary for Wnt5a-induced HS1 phosphorylation. Moreover, the cytoplamic, and more specifically the proline-rich domain (PRD), of ROR1 was required for it to associate with HS1 and allow for F-actin polymerization in response to Wnt5a. Accordingly, we introduced single amino acid substitutions of proline (P) to alanine (A) in the ROR1 PRD at positions 784, 808, 826, 841 or 850 in potential SH3-binding motifs. In contrast to wild-type ROR1, or other ROR1P→︀A mutants, ROR1P(841)A had impaired capacity to recruit HS1 and ARHGEF1 to ROR1 in response to Wnt5a. Moreover, Wnt5a could not induce cells expressing ROR1P(841)A to phosphorylate HS1 or activate ARHGEF1, and was unable to enhance CLL-cell motility. Collectively, these studies indicate HS1 plays an important role in ROR1-dependent Wnt5a-enhanced chemokine-directed leukemia-cell migration

Pre-M Phase-promoting Factor Associates with Annulate Lamellae in Xenopus Oocytes and Egg Extracts

We have used complementary biochemical and in vivo approaches to study the compartmentalization of M phase-promoting factor (MPF) in prophase Xenopus eggs and oocytes. We first examined the distribution of MPF (Cdc2/CyclinB2) and membranous organelles in high-speed extracts of Xenopus eggs made during mitotic prophase. These extracts were found to lack mitochondria, Golgi membranes, and most endoplasmic reticulum (ER) but to contain the bulk of the pre-MPF pool. This pre-MPF could be pelleted by further centrifugation along with components necessary to activate it. On activation, Cdc2/CyclinB2 moved into the soluble fraction. Electron microscopy and Western blot analysis showed that the pre-MPF pellet contained a specific ER subdomain comprising "annulate lamellae" (AL): stacked ER membranes highly enriched in nuclear pores. Colocalization of pre-MPF with AL was demonstrated by anti-CyclinB2 immunofluorescence in prophase oocytes, in which AL are positioned close to the vegetal surface. Green fluorescent protein-CyclinB2 expressed in oocytes also localized at AL. These data suggest that inactive MPF associates with nuclear envelope components just before activation. This association may explain why nuclei and centrosomes stimulate MPF activation and provide a mechanism for targeting of MPF to some of its key substrates

Computational Identification of Phospho-Tyrosine Sub-Networks Related to Acanthocyte Generation in Neuroacanthocytosis

Acanthocytes, abnormal thorny red blood cells (RBC), are one of the biological hallmarks of neuroacanthocytosis syndromes (NA), a group of rare hereditary neurodegenerative disorders. Since RBCs are easily accessible, the study of acanthocytes in NA may provide insights into potential mechanisms of neurodegeneration. Previous studies have shown that changes in RBC membrane protein phosphorylation state affect RBC membrane mechanical stability and morphology. Here, we coupled tyrosine-phosphoproteomic analysis to topological network analysis. We aimed to predict signaling sub-networks possibly involved in the generation of acanthocytes in patients affected by the two core NA disorders, namely McLeod syndrome (MLS, XK-related, Xk protein) and chorea-acanthocytosis (ChAc, VPS13A-related, chorein protein). The experimentally determined phosphoproteomic data-sets allowed us to relate the subsequent network analysis to the pathogenetic background. To reduce the network complexity, we combined several algorithms of topological network analysis including cluster determination by shortest path analysis, protein categorization based on centrality indexes, along with annotation-based node filtering. We first identified XK- and VPS13A-related protein-protein interaction networks by identifying all the interactomic shortest paths linking Xk and chorein to the corresponding set of proteins whose tyrosine phosphorylation was altered in patients. These networks include the most likely paths of functional influence of Xk and chorein on phosphorylated proteins. We further refined the analysis by extracting restricted sets of highly interacting signaling proteins representing a common molecular background bridging the generation of acanthocytes in MLS and ChAc. The final analysis pointed to a novel, very restricted, signaling module of 14 highly interconnected kinases, whose alteration is possibly involved in generation of acanthocytes in MLS and ChAc

Membrane association of peroxiredoxin-2 in red cells is mediated by the N-terminal cytoplasmic domain of band3

Band 3(B3),theaniontransporter,isanintegralmembraneproteinthatplaysakeystructuralroleby anchoringtheplasmamembranetothespectrin-basedmembraneskeletonintheredcell.Inaddition, it alsoplaysacriticalroleintheassemblyofglycolyticenzymestoregulateredcellmetabolism. However,itsabilitytorecruitproteinsthatcanpreventmembraneoxidationhasnotbeenpreviously explored.Inthisstudy,usingavarietyofexperimentalapproachesincludingcross-linkingstudies, fluorescenceanddichroicmeasurements,surfaceplasmonresonanceanalysis,andproteolyticdigestion assays,wedocumentthattheantioxidantproteinperoxiredoxin-2(PRDX2),thethirdmostabundant cytoplasmicproteininRBCs,interactswiththecytoplasmicdomainofB3.Thesurfaceelectrostatic potentialanalysisandstoichiometrymeasurementsrevealedthattheN-terminalpeptideofB3is involvedintheinteraction.PRDX2underwentaconformationalchangeuponitsbindingtoB3without losingitsperoxidaseactivity.HemichromeformationinducedbyphenylhydrazineofRBCsprevented membraneassociationofPRDX2,implyingoverlappingbindingsites.Documentationoftheabsenceof bindingofPRDX2toB3Neapolisredcellmembranes,inwhichtheinitialN-terminal11aminoacidsare deleted,enabledustoconcludethatPRDX2bindstotheN-terminalcytoplasmicdomainofB3andthat the first11aminoacidsofthisdomainarecrucialforPRDX2membraneassociationinintactRBCs. ThesefindingsimplyyetanotherimportantroleforB3inregulatingredcellmembranefunction

Membrane association of peroxiredoxin-2 in red cells is mediated by the N-terminal cytoplasmic domain of band 3

none12Band 3 (B3), the anion transporter, is an integral membrane protein that plays a key structural role by anchoring the plasma membrane to the spectrin-based membrane skeleton in the red cell. In addition, it also plays a critical role in the assembly of glycolytic enzymes to regulate red cell metabolism. However, its ability to recruit proteins that can prevent membrane oxidation has not been previously explored. In this study, using a variety of experimental approaches including cross-linking studies, fluorescence and dichroic measurements, surface plasmon resonance analysis, and proteolytic digestion assays, we document that the antioxidant protein peroxiredoxin-2 (PRDX2), the third most abundant cytoplasmic protein in RBCs, interacts with the cytoplasmic domain of B3. The surface electrostatic potential analysis and stoichiometry measurements revealed that the N-terminal peptide of B3 is involved in the interaction. PRDX2 underwent a conformational change upon its binding to B3 without losing its peroxidase activity. Hemichrome formation induced by phenylhydrazine of RBCs prevented membrane association of PRDX2, implying overlapping binding sites. Documentation of the absence of binding of PRDX2 to B3 Neapolis red cell membranes, in which the initial N-terminal 11 amino acids are deleted, enabled us to conclude that PRDX2 binds to the N-terminal cytoplasmic domain of B3 and that the first 11 amino acids of this domain are crucial for PRDX2 membrane association in intact RBCs. These findings imply yet another important role-for B3 in regulating red cell membrane functionnoneA. Matte;M. Bertoldi;N. Mohandas;X. An;A. Bugatti;A. M. Brunati;M. Rusnati;E. Tibaldi;A. Siciliano;F. Turrini;S. Perrotta;L. D. FranceschiA., Matte; M., Bertoldi; N., Mohandas; X., An; A., Bugatti; Brunati, ANNA MARIA; M., Rusnati; Tibaldi, Elena; A., Siciliano; F., Turrini; S., Perrotta; L. D., Francesch

Membrane association of peroxiredoxin-2 in red cells is mediated by n-terminal cytoplasmic domain of band 3.

Band-3 (B3), the anion transporter is an integral membrane protein that plays a key structural role by anchoring the plasma membrane to the spectrin-based membrane skeleton in the red cell. In addition, it also plays a critical role in the assembly of glycolytic enzymes to regulate red cell metabolism. However, its ability to recruit proteins that can prevent membrane oxidation has not been previously explored. In the present study, using a variety of experimental approaches including crosslinking studies, fluorescence and dichroic measurements, surface-plasmon-resonance analysis, and proteolytic digestion assays, we document that the anti-oxidant protein, Peroxiredoxin-2 (PRDX2), the third most abundant cytoplasmic protein in RBCs, interacts with the cytoplasmic domain of B3. The surface electrostatic potential analysis and stoichiometry measurements revealed that the N-terminal peptide of B3 is involved in interaction. PRDX2 underwent a conformational change upon its binding to B3 without losing its peroxidase activity. Hemichrome formation induced by phenylhydrazine (PHZ) of RBCs prevented membrane association of PRDX2 implying over lapping binding sites. Documentation of the absence of binding of PRDX2 to B3 Neapolis red cell membranes in which the initial N-terminal 11 amino acids are deleted, enabled us to conclude that PRDX2 binds to the N-terminal cytoplasmic domain of B3 and that the first 11 amino acid of this domain are crucial for PRDX2 membrane-association in intact RBCs. These findings imply yet another important role for B3 in regulating red cell membrane function