ASB proteins interact with Cullin5 and Rbx2 to form E3 ubiquitin ligase complexes

The ankyrin repeat and SOCS box (ASB) family is composed of 18 proteins from ASB1 to ASB18 and belongs to the suppressor of cytokine signaling (SOCS) box protein superfamily. ASB2 was recently shown to interact with a certain Cul-Rbx module to form an E3 ubiquitin (Ub) ligase complex, but the functional composition of the ASB-containing E3 Ub ligase complexes remains to be characterized. Here, we show that ASB proteins interact with Cul5-Rbx2 but neither Cul2 nor Rbx1 in cells. Mutational analysis revealed that the highly conserved amino acid sequences of the BC box and Cul5 box in the SOCS box of ASB proteins were essential for the interaction with Cul5-Rbx2. Although ASB proteins show slight divergences from the consensus sequences of the BC box and Cul5 box, all five tested ASB proteins bound to Cul5-Rbx2. Furthermore, all three tested ASB complexes containing Cul5-Rbx2 were found to have E3 Ub ligase activity. These findings suggest that the ASB family proteins interact with Cul5-Rbx2 to form E3 Ub ligases and play significant roles via a ubiquitination-mediated pathway.     

Calmodulin binds HER2 and modulates HER2 signaling

Human epidermal growth factor receptor 2 (HER2), a member of the ErbB family of receptor tyrosine kinases, has defined roles in neoplastic transformation and tumor progression. Overexpression of HER2 is an adverse prognostic factor in several human neoplasms and, particularly in breast cancer, correlates strongly with a decrease in overall patient survival. HER2 stimulates breast tumorigenesis by forming protein-protein interactions with a diverse array of intracellular signaling molecules, and evidence suggests that manipulation of these associations holds therapeutic potential. To modulate specific HER2 interactions, the region(s) of HER2 to which each target binds must be accurately identified. Calmodulin (CaM), a ubiquitously expressed Ca²⁺ binding protein, interacts with multiple intracellular targets. Interestingly, CaM binds the juxtamembrane region of the epidermal growth factor receptor, a HER2 homolog. Here, we show that CaM interacts, in a Ca²⁺-regulated manner, with two distinct sites on the N-terminal portion of the HER2 intracellular domain. Deletion of residues 676-689 and 714-732 from HER2 prevented CaM-HER2 binding. Inhibition of CaM function or deletion of the CaM binding sites from HER2 significantly decreased both HER2 phosphorylation and HER2-stimulated cell growth. Collectively, these data suggest that inhibition of CaM-HER2 interaction may represent a rational therapeutic strategy for the treatment of patients with breast cancer. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.     

SHIP2 associates with intersectin and recruits it to the plasma membrane in response to EGF

We identified intersectin1 (ITSN1) as a new binding partner of the SH2 domain containing inositol 5-phosphatase 2 (SHIP2). The interaction between SHIP2 and ITSN1 was confirmed in vivo. Src homology 3D, A, C, and E domains of ITSN1 were shown to be implicated in the interaction. In response to epidermal growth factor, SHIP2 expression could recruit the ITSN1 short form (ITSN1-S) to the cell membrane, while SHIP2 overexpression did not modulate the ITSN-mediated extracellular signal-regulated kinase1/2 and c-Jun NH2-terminal kinase activation. Our data provide a molecular link between SHIP2 and ITSN1 which are involved in receptor endocytosis regulation. STRUCTURED SUMMARY:     

Stabilin-1 and stabilin-2 are both directed into the early endocytic pathway in hepatic sinusoidal endothelium via interactions with clathrin/AP-2, independent of ligand binding

Liver sinusoidal endothelial cells (LSECs) mediate clearance of hyaluronan (HA) and scavenger receptor ligands, for example, advanced glycation end product (AGE)-modified proteins and oxidized lipids from the circulation. We recently cloned stabilin-1 and -2, two members of a novel family of transmembrane proteins expressed in LSECs. By using primary LSECs and HEK293 cells separately expressing either stabilin, we have investigated their roles in the early events of endocytosis with respect to localization, ligand-binding properties, and associations with clathrin and adaptor protein (AP)-2. Both stabilins were present at the cell surface, although surface levels of stabilin-1 were limited. In addition, stabilins were present in early endosomal antigen (EEA)-1+ organelles colocalizing with endocytosed AGE-modified bovine serum albumin (BSA). Treating cells with monensin further pronounced this distribution. Recombinant stabilin-2, but not recombinant stabilin-1, bound HA and the scavenger receptor ligands AGE-modified BSA, formaldehyde-treated BSA, and collagen N-terminal propeptides. In LSECs, both stabilins were associated with clathrin and AP-2, but not with each other. These interactions did not change upon addition of exogenous HA, suggesting that stabilins are constitutively internalized. In conclusion, hepatic stabilins are both present in the early endocytic pathway, associating with clathrin/AP-2, but whereas stabilin-2 has a clear scavenging profile, stabilin-1 does not recognize these ligands.     

Erythroid differentiation is augmented in Reelin-deficient K562 cells and homozygous reeler mice

Reelin is an extracellular glycoprotein that is highly conserved in mammals. In addition to its expression in the nervous system, Reelin is present in erythroid cells but its function there is unknown. We report in this study that Reelin is up-regulated during erythroid differentiation of human erythroleukemic K562 cells and is expressed in the erythroid progenitors of murine bone marrow. Reelin deficiency promotes erythroid differentiation of K562 cells and augments erythroid production in murine bone marrow. In accordance with these findings, Reelin deficiency attenuates AKT phosphorylation of the Ter119⁺CD71⁺ erythroid progenitors and alters the cell number and frequency of the progenitors at different erythroid differentiation stages. A regulatory role of Reelin in erythroid differentiation is thus defined.     

Myristoylation of human LanC-like Protein 2 (LANCL2) is essential for the interaction with the plasma membrane and the increase in cellular sensitivity to adriamycin

Human LANCL2, also known as Testis-specific Adriamycin Sensitivity Protein (TASP), is a member of the highly conserved and widely distributed lanthionine synthetase component C-like (LANCL) protein family. Expression studies of tagged LANCL2 revealed the major localization to the plasma membrane, juxta-nuclear vesicles, and the nucleus, in contrast to the homologue LANCL1 that was mainly found in the cytosol and nucleus. We identified the unique N-terminus of LANCL2 to function as the membrane anchor and characterized the relevant N-terminal myristoylation and a basic phosphatidylinositol phosphate-binding site. Interestingly, the non-myristoylated protein was confined to the nucleus indicating that the myristoylation targets LANCL2 to the plasma membrane. Cholesterol depletion by methyl-β-cyclodextrin caused the partial dissociation of overexpressed LANCL2 from the plasma membrane in vitro, whereas in vivo we observed an enhanced cell detachment from the matrix. We found that overexpressed LANCL2 interacts with the cortical actin cytoskeleton and therefore may play a role in cytoskeleton reorganization and in consequence to cell detachment. Moreover, we confirmed previous data that LANCL2 overexpression enhances the cellular sensitivity to the anticancer drug adriamycin and found that this sensitivity is dependent on the myristoylation and membrane association of LANCL2.     

Role of S-adenosylhomocysteine hydrolase in adenosine-induced apoptosis in HepG2 cells

Adenosine has been shown to initiate apoptosis through different mechanisms: (i) activation of adenosine receptors, (ii) intracellular conversion to AMP and stimulation of AMP-activated kinase, (iii) conversion to S-adenosylhomocysteine (AdoHcy), which is an inhibitor of S-adenosylmethionine (AdoMet)-dependent methyltransferases. Since the pathways involved are still not completely understood, we further investigated the role of AdoHcy hydrolase in adenosine-induced apoptosis. In HepG2 cells, adenosine induced caspase-like activity and DNA fragmentation, a marker of apoptosis. These effects were potentiated by co-incubation with homocysteine or adenosine deaminase inhibitor, pentostatin, and were mimicked by inhibition of AdoHcy hydrolase by adenosine-2',3'-dialdehyde (Adox). Adenosine-induced effects were significantly inhibited by dipyridamole, an inhibitor of adenosine transporter, whereas inhibitors of adenosine kinase did not affect adenosine-induced changes. Various adenosine receptor agonists and AICAR, an activator of AMP-activated kinase, did not mimic the effect of adenosine. Thus, adenosine-induced apoptosis is likely due to intracellular action of AdoHcy and independent of AMP-activated kinase and adenosine receptors. Because elevated AdoHcy levels are associated with reduced mRNA methylation, we studied mRNA expression in Adox-treated cells by microarray analysis. Since several p53-target genes and other apoptosis-related genes were up-regulated by Adox, we conclude that AdoHcy is involved in adenosine-induced apoptosis by altering gene expression.