PKCdelta acts as a growth and tumor suppressor in rat colonic epithelial cells.
(9/70987)
We have analysed the expression of three calcium-independent isoforms of protein kinase C (PKC), PKCdelta, PKCepsilon and PKCzeta, in an in vitro model of colon carcinogenesis consisting of the nontumorigenic rat colonic epithelial cell line D/WT, and a derivative src-transformed line D/src. While PKCzeta and PKCepsilon showed similar protein levels, PKCdelta was markedly decreased in D/src cells when compared to the D/WT line. To assess whether down-regulation of PKCdelta was causally involved in the neoplastic phenotype in D/src cells, we prepared a kinase-defective mutant of PKCdelta. Stable transfection of this sequence caused morphological and growth changes characteristic of partial transformation in D/WT cells. Moreover, to test whether PKCdelta was involved in growth control and transformation in this model, we overexpressed PKCdelta in D/src cells. Transfected cells underwent marked growth and morphological modifications toward the D/WT phenotype. In a late stage in culture, transfected cells ceased to proliferate, rounded up and degenerated into multinucleated, giant-like cells. We conclude that PKCdelta can reverse the transformed phenotype and act as a suppressor of cell growth in D/src cells. Moreover, our data show that downregulation of this isoenzyme of PKC may cooperate in the neoplastic transformation induced by the src oncogene in D/WT cells. (+info)
Phenotypic analysis of human glioma cells expressing the MMAC1 tumor suppressor phosphatase.
(10/70987)
MMAC1, also known as PTEN or TEP-1, was recently identified as a gene commonly mutated in a variety of human neoplasias. Sequence analysis revealed that MMAC1 harbored sequences similar to those found in several protein phosphatases. Subsequent studies demonstrated that MMAC1 possessed in vitro enzymatic activity similar to that exhibited by dual specificity phosphatases. To characterize the potential cellular functions of MMAC1, we expressed wild-type and several mutant variants of MMAC1 in the human glioma cell line, U373, that lacks endogenous expression. While expression of wild-type MMAC1 in these cells significantly reduced their growth rate and saturation density, expression of enzymatically inactive MMAC1 significantly enhanced growth in soft agar. Our observations indicate that while wild-type MMAC1 exhibits activities compatible with its proposed role as a tumor suppressor, cellular expression of MMAC1 containing mutations in the catalytic domain may yield protein products that enhance transformation characteristics. (+info)
Immune responses to all ErbB family receptors detectable in serum of cancer patients.
(11/70987)
Employing NIH3T3 transfectants with individual human ErbB receptor coding sequences as recombinant antigen sources, we detected by immunoblot analysis specific immunoreactivity against all four ErbB receptors among 13 of 41 sera obtained from patients with different types of epithelial malignancies. Overall, serum positivity was most frequently directed against ErbB2 followed by EGFR, ErbB3 and ErbB4. Specificity patterns comprised tumor patients with unique serum reactivity against ErbB2 or ErbB4. Moreover, approximately half of the positive sera exhibited concomitant reactivity with multiple ErbB receptors including EGFR and ErbB2, EGFR and ErbB4, ErbB2 and ErbB3 or EGFR, ErbB2 and ErbB3. Serum reactivity was confirmed for the respective ErbB receptors expressed by human tumor cells and corroborated on receptor-specific immunoprecipitates. Positive sera contained ErbB-specific antibodies of the IgG isotype. Representative immunohistochemical analysis of tumor tissues suggested overexpression of ErbB receptors for which serum antibodies were detectable in five of six patients. These findings implicate multiple ErbB receptors including ErbB3 and ErbB4 in addition to EGFR and ErbB2 in primary human cancer. Heterogeneity of natural ErbB-specific responses in cancer patients warrants their evaluation in light of immunotherapeutic approaches targeting these receptors. (+info)
Structural basis of profactor D activation: from a highly flexible zymogen to a novel self-inhibited serine protease, complement factor D.
(12/70987)
The crystal structure of profactor D, determined at 2.1 A resolution with an Rfree and an R-factor of 25.1 and 20.4%, respectively, displays highly flexible or disordered conformation for five regions: N-22, 71-76, 143-152, 187-193 and 215-223. A comparison with the structure of its mature serine protease, complement factor D, revealed major conformational changes in the similar regions. Comparisons with the zymogen-active enzyme pairs of chymotrypsinogen, trypsinogen and prethrombin-2 showed a similar distribution of the flexible regions. However, profactor D is the most flexible of the four, and its mature enzyme displays inactive, self-inhibited active site conformation. Examination of the surface properties of the N-terminus-binding pocket indicates that Ile16 may play the initial positioning role for the N-terminus, and Leu17 probably also helps in inducing the required conformational changes. This process, perhaps shared by most chymotrypsinogen-like zymogens, is followed by a factor D-unique step, the re-orientation of an external Arg218 to an internal position for salt-bridging with Asp189, leading to the generation of the self-inhibited factor D. (+info)
Binding of the G domains of laminin alpha1 and alpha2 chains and perlecan to heparin, sulfatides, alpha-dystroglycan and several extracellular matrix proteins.
(13/70987)
The C-terminal G domain of the mouse laminin alpha2 chain consists of five lamin-type G domain (LG) modules (alpha2LG1 to alpha2LG5) and was obtained as several recombinant fragments, corresponding to either individual modules or the tandem arrays alpha2LG1-3 and alpha2LG4-5. These fragments were compared with similar modules from the laminin alpha1 chain and from the C-terminal region of perlecan (PGV) in several binding studies. Major heparin-binding sites were located on the two tandem fragments and the individual alpha2LG1, alpha2LG3 and alpha2LG5 modules. The binding epitope on alpha2LG5 could be localized to a cluster of lysines by site-directed mutagenesis. In the alpha1 chain, however, strong heparin binding was found on alpha1LG4 and not on alpha1LG5. Binding to sulfatides correlated to heparin binding in most but not all cases. Fragments alpha2LG1-3 and alpha2LG4-5 also bound to fibulin-1, fibulin-2 and nidogen-2 with Kd = 13-150 nM. Both tandem fragments, but not the individual modules, bound strongly to alpha-dystroglycan and this interaction was abolished by EDTA but not by high concentrations of heparin and NaCl. The binding of perlecan fragment PGV to alpha-dystroglycan was even stronger and was also not sensitive to heparin. This demonstrated similar binding repertoires for the LG modules of three basement membrane proteins involved in cell-matrix interactions and supramolecular assembly. (+info)
Arrestin function in G protein-coupled receptor endocytosis requires phosphoinositide binding.
(14/70987)
Internalization of agonist-activated G protein-coupled receptors is mediated by non-visual arrestins, which also bind to clathrin and are therefore thought to act as adaptors in the endocytosis process. Phosphoinositides have been implicated in the regulation of intracellular receptor trafficking, and are known to bind to other coat components including AP-2, AP180 and COPI coatomer. Given these observations, we explored the possibility that phosphoinositides play a role in arrestin's function as an adaptor. High-affinity binding sites for phosphoinositides in beta-arrestin (arrestin2) and arrestin3 (beta-arrestin2) were identified, and dissimilar effects of phosphoinositide and inositol phosphate on arrestin interactions with clathrin and receptor were characterized. Alteration of three basic residues in arrestin3 abolished phosphoinositide binding with complete retention of clathrin and receptor binding. Unlike native protein, upon agonist activation, this mutant arrestin3 expressed in COS1 cells neither supported beta2-adrenergic receptor internalization nor did it concentrate in coated pits, although it was recruited to the plasma membrane. These findings indicate that phosphoinositide binding plays a critical regulatory role in delivery of the receptor-arrestin complex to coated pits, perhaps by providing, with activated receptor, a multi-point attachment of arrestin to the plasma membrane. (+info)
The splicing factor-associated protein, p32, regulates RNA splicing by inhibiting ASF/SF2 RNA binding and phosphorylation.
(15/70987)
The cellular protein p32 was isolated originally as a protein tightly associated with the essential splicing factor ASF/SF2 during its purification from HeLa cells. ASF/SF2 is a member of the SR family of splicing factors, which stimulate constitutive splicing and regulate alternative RNA splicing in a positive or negative fashion, depending on where on the pre-mRNA they bind. Here we present evidence that p32 interacts with ASF/SF2 and SRp30c, another member of the SR protein family. We further show that p32 inhibits ASF/SF2 function as both a splicing enhancer and splicing repressor protein by preventing stable ASF/SF2 interaction with RNA, but p32 does not block SRp30c function. ASF/SF2 is highly phosphorylated in vivo, a modification required for stable RNA binding and protein-protein interaction during spliceosome formation, and this phosphorylation, either through HeLa nuclear extracts or through specific SR protein kinases, is inhibited by p32. Our results suggest that p32 functions as an ASF/SF2 inhibitory factor, regulating ASF/SF2 RNA binding and phosphorylation. These findings place p32 into a new group of proteins that control RNA splicing by sequestering an essential RNA splicing factor into an inhibitory complex. (+info)
Systemic administration of rIL-12 synergistically enhances the therapeutic effect of a TNF gene-transduced cancer vaccine.
(16/70987)
Interleukin-12 (IL-12) is a potent antitumor cytokine, which induces and enhances the activity of natural killer (NK) cells, lymphokine activated killer (LAK) cells and cytotoxic T lymphocytes (CTL). IL-12 also stimulates IFN-gamma production from both T cells and NK cells. In this study, we transfected methylcholanthrene-induced fibrosarcoma (MCA-D) with TNF gene and investigated the therapeutic effect of TNF gene-transduced cancer vaccine and whether the vaccination effect is enhanced by systemic administration of recombinant IL-12 (rIL-12), in a murine model. TNF gene-transduced cancer vaccine or systemic administration of rIL-12 showed slight or moderate inhibition of pre-established tumor. However, simultaneous application of the vaccine and rIL-12 resulted in complete eradication. The cytotoxicity of CTL against parental tumor cells was enhanced with the combination of the vaccine and rIL-12, and IFN-gamma production from spleen cells also increased synergistically. Our findings show that synergistic enhancement of CTL activity and IFN-gamma production could play an important role in the antitumor effect of combination therapy using TNF gene-transduced cancer vaccine and rIL-12. (+info)