CPTP1 is a nontransmembrane chicken protein tyrosine phosphatase having 92% sequence homology to the corresponding 321 amino acids of human protein tyrosine phosphatase 1B (HPTP1B). Using anti-CPTP1 antibody, we identified CPTP1-like rat PTP1 of 51 kappa Da in Rat-1 and v-src-transformed Rat-1 fibroblasts. Here we show that CPTP1-like rat PTP1 binds to p60v-src in vivo and CPTP1 also can associate with p60v-src in cell lysate of v-src- transformed Rat-1 fibroblasts. Interaction between HPTP1B-type PTPs, CPTP1-like rat PTP1 and CPTP1, and p60v-src was reduced by vanadate treatment for 13 h due to down regulation of the protein level of p60v-src in vivo. Interestingly, CPTP1-like rat PTP1 was coimmunoprecipitated with a 70-kappa Da protein which has a possibility to be tyrosine- phosphorylated by p60v-src in v-src-transformed Rat- 1 fibroblasts. These results suggest that HPTP1B- type PTPs may play an important role in p60src dependent signal pathway in eucaryotic cells.
Animals ; Blotting, Western ; Cell Line, Transformed ; Chickens ; Female ; Fibroblasts ; Oncogene Protein pp60(v-src)/*metabolism ; Phosphoprotein Phosphatase/genetics/*metabolism ; Precipitin Tests ; Protein Binding ; Protein-Tyrosine-Phosphatase/genetics/*metabolism ; Rabbits ; Rats ; Recombinant Fusion Proteins/genetics/metabolism

OBJECTIVEThe protozoan Toxoplasma gondii expresses large amounts of a 37 kDa Type 2C serine-threonine phosphatase, the so-called TgPP2C which has been suggested to contribute to parasite growth regulation. Ectopic expression in mammalian cells also indicated that the enzyme could regulate growth and survival. In this study, we aimed to investigate the interaction of TgPP2C with human SSRP1 (structure-specific recognition protein 1) and the effects of TgPP2C on cell viability.

METHODSThe yeast two hybrid system, His-tag pull-down and co-immunoprecipitation assays were used to confirm the interaction of TgPP2C with SSRP1 and determine the binding domain on SSRP1. The evaluation of cell apoptosis was performed using cleaved caspase-3 antibody and Annexin-V/PI kit combined with flow cytometry.

RESULTSWe identified human SSRP1 as an interacting partner of TgPP2C. The C-terminal region of SSRP1 including the amino acids 471 to 538 was specifically mapped as the region responsible for interaction with TgPP2C. The overexpression of TgPP2C down-regulated cell apoptosis and negatively regulated apoptosis induced by DRB, casein kinase II (CKII) inhibitor, through enhanced interaction with SSRP1.

CONCLUSIONTgPP2C may be a parasitic factor capable of promoting cell survival through interaction with the host protein SSRP1, thereby creating a favorable environment for parasite growth.

Apoptosis ; Blotting, Western ; DNA-Binding Proteins ; genetics ; metabolism ; Flow Cytometry ; HeLa Cells ; High Mobility Group Proteins ; genetics ; metabolism ; Humans ; Immunoprecipitation ; Phosphoprotein Phosphatases ; genetics ; metabolism ; Protein Phosphatase 2C ; Toxoplasma ; enzymology ; Transcriptional Elongation Factors ; genetics ; metabolism ; Two-Hybrid System Techniques

A previous report by this laboratory demonstrated that bacterial iron chelator (siderophore) triggers inflammatory signals, including the production of CXC chemokine IL-8, in human intestinal epithelial cells (IECs). Microarray-based gene expression profiling revealed that iron chelator also induces macrophage inflammatory protein 3 alpha (MIP-3alpha)/ CC chemokine-ligand 20 (CCL20). As CCL20 is chemotactic for the cells involved in host adaptive immunity, this suggests that iron chelator may stimulate IECs to have the capacity to link mucosal innate and adaptive immunity. The basal medium from iron chelator deferoxamine (DFO)-treated HT-29 monolayers was as chemotactic as recombinant human CCL20 at equivalent concentrations to attract CCR6+ cells. The increase of CCL20 protein secretion appeared to correspond to that of CCL20 mRNA levels, as determined by real-time quantitative RT-PCR. The efficacy of DFO at inducing CCL20 mRNA was also observed in human PBMCs and in THP-1 cells, but not in human umbilical vein endothelial cells. Interestingly, unlike other proinflammatory cytokines, such as TNF-alpha and IL-1beta, a time-dependent experiment revealed that DFO slowly induces CCL20, suggesting a novel mechanism of action. A pharmacologic study also revealed that multiple signaling pathways are differentially involved in CCL20 production by DFO, while some of those pathways are not involved in TNF-alpha-induced CCL20 production. Collectively, these results demonstrate that, in addition to some bacterial products known to induce host adaptive immune responses, direct chelation of host iron by infected bacteria may also contribute to the initiation of host adaptive immunity in the intestinal mucosa.
Calcium/metabolism ; Cell Movement/drug effects ; Chemokines, CC/genetics/*metabolism ; Deferoxamine/*pharmacology ; Egtazic Acid/analogs & derivatives/pharmacology ; HT29 Cells ; Humans ; Immunity, Mucosal/*drug effects ; Intestinal Mucosa/*drug effects/immunology/metabolism ; Iron Chelating Agents/*pharmacology ; Macrophage Inflammatory Proteins/genetics/*metabolism ; NF-kappa B/metabolism ; Phosphoprotein Phosphatase/physiology ; Protein Transport/drug effects ; Protein-Serine-Threonine Kinases/physiology ; RNA, Messenger/genetics/metabolism ; Receptors, Chemokine/metabolism ; Research Support, Non-U.S. Gov't