Mikhail A. Kutuzov

Cadherin 13 in cancer.

We review the evidence suggesting the involvement of Cadherin 13 (CDH13, T‐cadherin, H‐cadherin) in various cancers. CDH13 is an atypical member of the cadherin family, devoid of a transmembrane domain and anchored to the exterior surface of the plasma membrane via a glycosylphosphatidylinositol anchor. CDH13 is thought to affect cellular behavior largely through its signaling properties. It is often down‐regulated in cancerous cells. CDH13 down‐regulation has been associated with poorer prognosis in various carcinomas, such as lung, ovarian, cervical and prostate cancer. CDH13 re‐expression in most cancer cell lines inhibits cell proliferation and invasiveness, increases susceptibility to apoptosis, and reduces tumor growth in in vivo models. These properties suggest that CDH13 may represent a possible target for therapy in some cancers. At the same time, CDH13 is up‐regulated in blood vessels growing through tumors and promotes tumor neovascularization. In contrast to most cancer cell lines, CDH13 overexpression in endothelial cells promotes their proliferation and migration, and has a pro‐survival effect. We also discuss molecular mechanisms that may regulate CDH13 expression and underlie its roles in cancer.

Protozoan protein tyrosine phosphatases

The aim of this review is to provide a synthesis of the published experimental data on protein tyrosine phosphatases from parasitic protozoa, in silico analysis based on the availability of completed genomes and to place available data for individual phosphatases from different unicellular parasites into the comparative and evolutionary context. We analysed the complement of protein tyrosine phosphatases (PTP) in several species of unicellular parasites that belong to Apicomplexa (Plasmodium; Cryptosporidium, Babesia, Theileria, and Toxoplasma), kinetoplastids (Leishmania and Trypanosoma spp.), as well as Entamoeba histolytica, Giardia lamblia, Trichomonas vaginalis and a microsporidium Encephalitozoon cuniculi. The analysis shows distinct distribution of the known families of tyrosine phosphatases in different species. Protozoan tyrosine phosphatases show considerable levels of divergence compared with their mammalian homologues, both in terms of sequence similarity between the catalytic domains and the structure of their flanking domains. This potentially makes them suitable targets for development of specific inhibitors with minimal effects on physiology of mammalian hosts.

Gα12 is targeted to the mitochondria and affects mitochondrial morphology and motility

Gα12 constitutes, along with Gα13, one of the four families of α subunits of heterotrimeric G proteins. We found that the N terminus of Gα12, but not those of other Gα subunits, contains a predicted mitochondrial targeting sequence. Using confocal microscopy and cell fractionation, we demonstrated that up to 40% of endogenous Gα12 in human umbilical vein endothelial cells colocalize with mitochondrial markers. N‐terminal sequence of Gα12 fused to GFP efficiently targeted the fusion protein to mitochondria. Gα12 with mutated mitochondrial targeting sequence was still located in mitochondria, suggesting the existence of additional mechanisms for mitochondrial localization. Lysophosphatidic acid, one of the known stimuli transduced by Gα12/13, inhibited mitochondrial motility, while depletion of endogenous Gα12 increased mitochondrial motility. Gα12Q229L variants uncoupled from RhoGEFs (but not fully functional activated Gα12Q229L) induced transformation of the mitochondrial network into punctate mitochondria and resulted in a loss of mitochondrial membrane potential. All examined Gα12Q229L variants reduced phosphorylation of Bcl‐2 at Ser‐70, while only mutants unable to bind RhoGEFs also decreased cellular levels of Bcl‐2. These Gα12 mutants were also more efficient Hsp90 interactors. These findings are the first demonstration of a heterotrimeric G protein α subunit specifically targeted to mitochondria and involved in the control of mitochondrial morphology and dynamics.—Andreeva, A. V., Kutuzov, M. A., Voyno‐Yasenetskaya, T. A. Gα12 is targeted to the mitochondria and affects mitochondrial morphology and motility. FASEB J. 22, 2821–2831 (2008)

Gα12 Interaction with αSNAP Induces VE-cadherin Localization at Endothelial Junctions and Regulates Barrier Function

The involvement of heterotrimeric G proteins in the regulation of adherens junction function is unclear. We identified αSNAP as an interactive partner of Gα12 using yeast two-hybrid screening. glutathione S-transferase pull-down assays showed the selective interaction of αSNAP with Gα12 in COS-7 as well as in human umbilical vein endothelial cells. Using domain swapping experiments, we demonstrated that the N-terminal region of Gα12 (1–37 amino acids) was necessary and sufficient for its interaction with αSNAP. Gα13 with its N-terminal extension replaced by that of Gα12 acquired the ability to bind to αSNAP, whereas Gα12 with its N terminus replaced by that of Gα13 lost this ability. Using four point mutants of αSNAP, which alter its ability to bind to the SNARE complex, we determined that the convex rather than the concave surface of αSNAP was involved in its interaction with Gα12. Co-transfection of human umbilical vein endothelial cells with Gα12 and αSNAP stabilized VE-cadherin at the plasma membrane, whereas down-regulation of αSNAP with siRNA resulted in the loss of VE-cadherin from the cell surface and, when used in conjunction with Gα12 overexpression, decreased endothelial barrier function. Our results demonstrate a direct link between the α subunit of G12 and αSNAP, an essential component of the membrane fusion machinery, and implicate a role for this interaction in regulating the membrane localization of VE-cadherin and endothelial barrier function.

A ubiquitous membrane fusion protein αSNAP: a potential therapeutic target for cancer, diabetes and neurological disorders?

Alpha soluble NSF attachment protein (αSNAP) is a ubiquitous and indispensable component of membrane fusion machinery. Deletion of αSNAP is embryonically lethal. Yet, there is accumulating evidence that milder alterations in expression levels of αSNAP may be associated with a number of specific pathological conditions, such as several neurological disorders, Type 2 diabetes and aggressive neuroendocrine tumours. Here, the authors review the evidence available for animal models and for humans, and discuss possible therapeutic approaches that may target αSNAP.

T-cadherin modulates endothelial barrier function.

T‐cadherin is an atypical member of the cadherin family, which lacks the transmembrane and intracellular domains and is attached to the plasma membrane via a glycosylphosphatidylinositol anchor. Unlike canonical cadherins, it is believed to function primarily as a signaling molecule. T‐cadherin is highly expressed in endothelium. Using transendothelial electrical resistance measurements and siRNA‐mediated depletion of T‐cadherin in human umbilical vein endothelial cells, we examined its involvement in regulation of endothelial barrier. We found that in resting confluent monolayers adjusted either to 1% or 10% serum, T‐cadherin depletion modestly, but consistently reduced transendothelial resistance. This was accompanied by increased phosphorylation of Akt and LIM kinase, reduced phosphorylation of p38 MAP kinase, but no difference in tubulin acetylation and in phosphorylation of an actin filament severing protein cofilin and myosin light chain kinase. Serum stimulation elicited a biphasic increase in resistance with peaks at 0.5 and 4–5 h, which was suppressed by a PI3 kinase/Akt inhibitor wortmannin and a p38 inhibitor SB 239063. T‐cadherin depletion increased transendothelial resistance between the two peaks and reduced the amplitude of the second peak. T‐cadherin depletion abrogated serum‐induced Akt phosphorylation at Thr308 and reduced phosphorylation at Ser473, reduced phosphorylation of cofilin, and accelerated tubulin deacetylation. Adiponectin slightly improved transendothelial resistance irrespectively of T‐cadherin depletion. T‐cadherin depletion also resulted in a reduced sensitivity and delayed responses to thrombin. These data implicate T‐cadherin in regulation of endothelial barrier function, and suggest a complex signaling network that links T‐cadherin and regulation of barrier function. J. Cell. Physiol. 223: 94–102, 2010.

T-cadherin is located in the nucleus and centrosomes in endothelial cells

T-cadherin (H-cadherin, cadherin 13) is upregulated in vascular proliferative disorders and in tumor-associated neovascularization and is deregulated in many cancers. Unlike canonical cadherins, it lacks transmembrane and intracellular domains and is attached to the plasma membrane via a glycosylphosphatidylinositol anchor. T-cadherin is thought to function in signaling rather than as an adhesion molecule. Some interactive partners of T-cadherin at the plasma membrane have recently been identified. We examined T-cadherin location in human endothelial cells using confocal microscopy and subcellular fractionation. We found that a considerable proportion of T-cadherin is located in the nucleus and in the centrosomes. T-cadherin colocalized with a centrosomal marker gamma-tubulin uniformly throughout the cell cycle at least in human umbilical vein endothelial cells. In the telophase, T-cadherin transiently concentrated in the midbody and was apparently degraded. Its overexpression resulted in an increase in the number of multinuclear cells, whereas its downregulation by small interfering RNA led to an increase in the number of cells with multiple centrosomes. These findings indicate that deregulation of T-cadherin in endothelial cells may lead to disturbances in cytokinesis or centrosomal replication.

Protein phosphatase with EF-hand domains 2 (PPEF2) is a potent negative regulator of apoptosis signal regulating kinase-1 (ASK1).

The function of protein phosphatases with EF-hand domains (PPEF) in mammals is not known. Large-scale expression profiling experiments suggest that PPEF expression may correlate with stress protective responses, cell survival, growth, proliferation, or neoplastic transformation. Apoptosis signal regulating kinase-1 (ASK1) is a MAP kinase kinase kinase implicated in cancer, cardiovascular and neurodegenerative diseases. ASK1 is activated by oxidative stress and induces pro-apoptotic or inflammatory signalling, largely via sustained activation of MAP kinases p38 and/or JNK. We identify human PPEF2 as a novel interacting partner and a negative regulator of ASK1. In COS-7 or HEK 293A cells treated with H(2)O(2), expression of PPEF2 abrogated sustained activation of p38 and one of the JNK p46 isoforms, and prevented ASK1-dependent caspase-3 cleavage and activation. PPEF2 efficiently suppressed H(2)O(2)-induced activation of ASK1. Overexpessed as well as endogenous ASK1 co-immunoprecipitated with PPEF2. PPEF2 was considerably more potent both as a suppressor of ASK1 activation and as its interacting partner as compared to protein phosphatase 5 (PP5), a well-known negative regulator of ASK1. PPEF2 was found to form complexes with endogenous Hsp70 and to a lesser extent Hsp90, which are also known interacting partners of PP5. These data identify, for the first time, a possible downstream signalling partner of a mammalian PPEF phosphatase, and suggest that, despite structural divergence, PPEF and PP5 phosphatases may share common interacting partners and functions.

Regulation of apoptosis signal-regulating kinase 1 degradation by Gα13

Apoptosis signal‐regulating kinase (ASK1) is a mitogen‐activated protein kinase (MAPK) that transduces apoptotic signals from a variety of stresses. We have shown previously that alpha subunits of heterotrimeric G12 and G13 proteins stimulate ASK1 kinase activity and ASK1‐dependent apoptosis (1). Here, we report a novel mechanism of G‐protein‐dependent regulation of ASK1. We demonstrated that Go 13 forms a complex with ASK1 in an activation‐independent manner. Both N‐ and C‐terminal regulatory domains of ASK1 were essential for the efficient interaction, while its kinase domain was not required. Formation of the Gα13‐ASK1 complex was enhanced by JNK‐interacting leucine zipper protein, JLP. Constitutively activated Gα13Q226L increased ASK1 expression. Short‐term activation of a serotonin 5‐HT4 receptor that is coupled to Gα13 also increased ASK1 expression. Importantly, prolonged activation of 5‐HT4 receptor in COS‐7 cells or prolonged treatment of human umbilical vein endothelial cells with thrombin concomi‐tantly down‐regulated both Gα13 and ASK1. Data showed that Gα13Q226L reduced the rate of ASK1 degradation, decreased ASK1 ubiquitination, and reduced association of ASK1 with an E3 ubiquitin ligase CHIP, previously shown to mediate ASK1 degradation. Our findings indicate that ASK1 expression levels can be regulated by Gα13, at least in part via control of ASK1 ubiquitination and degradation.—Kutuzov, M. A., Andreeva, A. V., Voyno‐Yasenetskaya, T. A. Regulation of apoptosis signal‐regulating kinase 1 degradation by Gα13. FASEB J. 21, 3727–3736 (2007)

Scaffolding proteins in G-protein signaling

Heterotrimeric G proteins are ubiquitous signaling partners of seven transmembrane-domain G-protein-coupled receptors (GPCRs), the largest (and most important pharmacologically) receptor family in mammals. A number of scaffolding proteins have been identified that regulate various facets of GPCR signaling. In this review, we summarize current knowledge concerning those scaffolding proteins that are known to directly bind heterotrimeric G proteins, and discuss the composition of the protein complexes they assemble and their effects on signal transduction. Emerging evidence about possible ways of regulation of activity of these scaffolding proteins is also discussed.