Alexandra V. Andreeva

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.

Endomembranes and vesicle trafficking.

Over the past year extensive analyses of the accumulated data on the structural and functional organisation of the endomembrane system and vesicular trafficking in higher plants have shown it to be far more complex than previously anticipated. The availability of molecular tools combined with new opportunities to visualise endomembrane dynamics in vivo will allow better understanding of the fundamental processes underlying the complexity of endomembrane behaviour and vesicular trafficking.

Organization of transport from endoplasmic reticulum to Golgi in higher plants

In plant cells, the organization of the Golgi apparatus and its interrelationships with the endoplasmic reticulum differ from those in mammalian and yeast cells. Endoplasmic reticulum and Golgi apparatus can now be visualized in plant cells in vivo with green fluorescent protein (GFP) specifically directed to these compartments. This makes it possible to study the dynamics of the membrane transport between these two organelles in the living cells. The GFP approach, in conjunction with a considerable volume of data about proteins participating in the transport between endoplasmic reticulum and Golgi in yeast and mammalian cells and the identification of their putative plant homologues, should allow the establishment of an experimental model in which to test the involvement of the candidate proteins in plants. As a first step towards the development of such a system, we are using Sar1, a small G-protein necessary for vesicle budding from the endoplasmic reticulum. This work has demonstrated that the introduction of Sar1 mutants blocks the transport from endoplasmic reticulum to Golgi in vivo in tobacco leaf epidermal cells and has therefore confirmed the feasibility of this approach to test the function of other proteins that are presumably involved in this step of endomembrane trafficking in plant cells.

RdgC/PP5-related phosphatases: novel components in signal transduction.

A great variety of cellular functions are regulated by protein serine/threonine phosphatases (PP). This review summarises the current knowledge of the structural features, patterns of expression and involvement in signal transduction pathways of protein serine/threonine phosphatases related to PP5 and RdgC. Designated now as PP5/RdgC subfamily by P. T. W. Cohen in her 1997 study published in Trends in Biochemical Sciences, (Vol. 22, pp. 245-251), this heterogeneous group comprises phosphatases PP5/PPT, containing regulatory domains with tetratricopeptide repeats, RdgC/PPEF, which possess Ca2+-binding EF hand-type sites, and, recently discovered in plants, PP7. PP5 is ubiquitously expressed and appears to be a multifunctional phosphatase involved in a number of different signalling pathways. In contrast, expression of RdgC/PPEF phosphatases and PP7 is confined primarily to specialised sensory cells in animals and plants, respectively, which may be indicative of their more specialised roles in sensory signal transduction.

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.

Cytoplasmic illuminations: in planta targeting of fluorescent proteins to cellular organelles.

SummaryUse of the jellyfish green-fluorescent protein as an in vivo reporter is in the process of revolutionising plant cell biology. By fusing the protein to specific targeting peptides or to sequences of complete proteins, it is now possible to observe the location, structure, and dynamics of a number of intracellular organelles over extended periods of time. In this review we discuss the most recent developments and unexpected results originating from the targeting of this unique protein and its derivatives to elements of the cytoskeleton and to membrane-bounded organelles in a range of plant cell types.

Expression and characterization of PP7, a novel plant protein Ser/Thr phosphatase distantly related to RdgC/PPEF and PP5

We have recently identified an Arabidopsis thaliana cDNA encoding a putative protein Ser/Thr phosphatase PP7, not closely related to any protein phosphatases in animals or fungi. Here, we describe the characterization of PP7 expressed in a bacterial system. The recombinant protein was inactive unless the longest insert in its catalytic domain was cleaved, suggesting that this insert is an autoinhibitory region. PP7 was resistant to okadaic acid, calyculin and fumonisin B1, and was stimulated by Mn2+ or Fe2+, while Ni2+ and Zn2+ were inhibitory. Polylysine stimulated PP7 activity towards p‐nitrophenylphosphate but inhibited activity towards the most efficient protein substrate, myelin basic protein. A tentative model of the control of PP7 activity is proposed.

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)

Protein Ser/Thr phosphatases with kelch-like repeat domains

This report describes the presence in plants of protein Ser/Thr phosphatases of the PPP family, homologous to PfPPalpha phosphatase from Plasmodium falciparum. Like PfPPalpha, they possess large N-terminal domains and catalytic domains that are more closely related to the protein phosphatase 1 group. The N-terminal domains of PfPPalpha and its plant homologues contain tandem kelch-like repeats, not previously identified in any protein phosphatases, suggesting that the N-terminal domains may form beta-propeller structures mediating protein-protein interactions. We therefore suggest that this novel phosphatase group be designated as PPKLs for protein phosphatases with kelch-like repeat domains. Four PPKL isoforms are encoded in the Arabidopsis thaliana genome, of which at least three are expressed. PPKLs appear to be ubiquitous in Viridiplantae. The existence of a protein phosphatase group shared by Viridiplantae and Apicomplexa, but not other eukaryotes, is in line with the theory of the origin of Apicomplexa by endosymbiosis of nonphotosynthetic eukaryotes with red algae.

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.