Actin filament-associated protein 1 is required for cSrc activity and secretory activation in the lactating mammary gland.

Actin filament-associated protein 1 (AFAP1) is an adaptor protein of cSrc that binds to filamentous actin and regulates the activity of this tyrosine kinase to affect changes to the organization of the actin cytoskeleton. In breast and prostate cancer cells, AFAP1 has been shown to regulate cellular responses requiring actin cytoskeletal changes such as adhesion, invadopodia formation and invasion. However, a normal physiologic role for AFAP1 has remained elusive. In this study, we generated an AFAP1 knockout mouse model that establishes a novel physiologic role for AFAP1 in lactation. Specifically, these animals displayed a defect in lactation that resulted in an inability to nurse efficiently. Histologically, the mammary glands of the lactating knockout mice were distinguished by the accumulation of large cytoplasmic lipid droplets in the alveolar epithelial cells. There was a reduction in lipid synthesis and the expression of lipogenic genes without a corresponding reduction in the production of ß-casein, a milk protein. Furthermore, these defects were associated with histologic and biochemical signs of precocious involution. This study also demonstrated that AFAP1 responds to prolactin, a lactogenic hormone, by forming a complex with cSrc and becoming tyrosine phosphorylated. Taken together, these observations pointed to a defect in secretory activation. Certain characteristics of this phenotype mirrored the defect in secretory activation in the cSrc knockout mouse, but most importantly, the activity of cSrc in the mammary gland was reduced during early lactation in the AFAP1-null mouse and the localization of active cSrc at the apical surface of luminal epithelial cells during lactation was selectively lost in the absence of AFAP1. These data define, for the first time, the requirement of AFAP1 for the spatial and temporal regulation of cSrc activity in the normal breast, specifically for milk production.

SRChing for the substrates of Src.

By the mid 1980's, it was clear that the transforming activity of oncogenic Src was linked to the activity of its tyrosine kinase domain and attention turned to identifying substrates, the putative next level of control in the pathway to transformation. Among the first to recognize the potential of phosphotyrosine-specific antibodies, Parsons and colleagues launched a risky shotgun-based approach that led ultimately to the cDNA cloning and functional characterization of many of today's best-known Src substrates (for example, p85-Cortactin, p110-AFAP1, p130Cas, p125FAK and p120-catenin). Two decades and over 6000 citations later, the original goals of the project may be seen as secondary to the enormous impact of these protein substrates in many areas of biology. At the request of the editors, this review is not restricted to the current status of the substrates, but reflects also on the anatomy of the project itself and some of the challenges and decisions encountered along the way.

Individualized survival and treatment response predictions for breast cancers using phospho-EGFR, phospho-ER, phospho-HER2/neu, phospho-IGF-IR/In, phospho-MAPK, and phospho-p70S6K proteins.

The development and progression of breast cancer involves the activation of numerous protein kinases, and the change in phosphorylation is a hallmark of protein kinase activation. In this study, we identified a comprehensive profile to predict individual breast cancer patients' survival and treatment responses using the Random Committee algorithm. The profile incorporated a subset of phosphorylated signal protein expressions and several selected clinical factors of breast cancer. The parameters of our profile were identified by supervised feature selection algorithms, Gain Ratio Attribute Evaluation and Relief. The results showed that the overall accuracy of survival prediction reached 92.3% for individual breast cancer patients with the use of the expression profiles of phospho-EGFR, phospho-ER, phospho-HER2/neu, phospho-IGFIR/In, phospho-MAPK, and phospho-p70S6K plus the selected clinical factors. The results also indicated that the overall accuracy of treatment response prediction was 92.6% with the use of the level of phospho-EGFR, phospho-ER, phospho-HER2/neu, phospho-MAPK, and phospho-p70S6K plus the selected clinical information. The prediction system combines multiple signal protein activation profiles and relevant clinical information, and provides a unique guideline to aid individualized decision-making in the clinical management of breast cancer.

Antitumor effect of beta-elemene in non-small-cell lung cancer cells is mediated via induction of cell cycle arrest and apoptotic cell death.

Beta-elemene is a novel anticancer drug, which was extracted from the ginger plant. However, the mechanism of action of beta-elemene in non-small-cell lung cancer (NSCLC) remains unknown. Here we show that beta-elemene had differential inhibitory effects on cell growth between NSCLC cell lines and lung fibroblast and bronchial epithelial cell lines. In addition, beta-elemene was found to arrest NSCLC cells at G2-M phase, the arrest being accompanied by decreases in the levels of cyclin B1 and phospho-Cdc2 (Thr-161) and increases in the levels of p27(kip1) and phospho-Cdc2 (Tyr-15). Moreover, beta-elemene reduced the expression of Cdc25C, which dephosphorylates/activates Cdc2, but enhanced the expression of the checkpoint kinase, Chk2, which phosphorylates/ inactivates Cdc25C. These findings suggest that the effect of beta-elemene on G2-M arrest in NSCLC cells is mediated partly by a Chk2-dependent mechanism. We also demonstrate that beta-elemene triggered apoptosis in NSCLC cells. Our results clearly show that beta-elemene induced caspase-3, -7 and -9 activities, decreased Bcl-2 expression, caused cytochrome c release and increased the levels of cleaved caspase-9 and poly(ADP-ribose) polymerase in NSCLC cells. These data indicate that the effect of beta-elemene on lung cancer cell death may be through a mitochondrial release of the cytochrome c-mediated apoptotic pathway.

Antiproliferative effect of beta-elemene in chemoresistant ovarian carcinoma cells is mediated through arrest of the cell cycle at the G2-M phase.

Elemene is a natural antitumor plant drug. However, the effect of elemene on cell growth in ovarian cancer is unknown. In this study, we show that beta-elemene inhibited the proliferation of cisplatin-resistant human ovarian cancer cells and their parental cells, but had only a marginal effect in human ovary cells, indicating differential inhibitory effects on cell growth between ovarian cancer cells and normal ovary cells. We also demonstrated for the first time that beta-elemene markedly enhanced cisplatin-induced growth inhibition in resistant cells compared to sensitive cells. In addition, cell cycle analysis revealed a synergistic effect of beta-elemene and cisplatin on the induction of cell cycle G2-M arrest in our resistant ovarian carcinoma cells. Furthermore, we showed that treatment of these cells with both drugs downregulated cyclin B1 and Cdc2 expression, but elevated the levels of p53, p21waf1/cip1, p27kip1 and Gadd45. Finally, the combination of beta-elemene and cisplatin was found to increase the phosphorylation of Cdc2 and Cdc25C, which leads to a reduction in Cdc2-cyclin B1 activity. These novel findings suggest that beta-elemene sensitizes chemoresistant ovarian carcinoma cells to cisplatin-induced growth suppression partly through modulating the cell cycle G2 checkpoint and inducing cell cycle G2-M arrest, which lead to blockade of cell cycle progression.

The intrinsic ability of AFAP-110 to alter actin filament integrity is linked with its ability to also activate cellular tyrosine kinases.

The actin filament-associated protein of 110 kDa (AFAP-110) is a Src binding partner that represents a potential modulator of actin filament integrity in response to cellular signals. Previous reports have demonstrated that AFAP-110 is capable of directly binding and altering actin filaments. Deletion of the leucine zipper motif of AFAP-110 (AFAP-110(Deltalzip)) has been shown to induce a phenotype which resembles Src-transformed cells, by repositioning actin filaments into rosettes. This deletion also mimics a conformational change in AFAP-110 that is detected in Src-transformed cells. The results presented here indicate that unlike AFAP-110, AFAP-110(Deltalzip) is capable of activating cellular tyrosine kinases, including Src family members, and that AFAP-110(Deltalzip) itself is hyperphosphorylated. The newly tyrosine phosphorylated proteins and activated Src-family members appear to be associated with actin-rich lamellipodia. A point mutation that alters the SH3-binding motif of AFAP-110(Deltalzip) prevents it from activating tyrosine kinases and altering actin filament integrity. In addition, a deletion within a pleckstrin homology (PH) domain of AFAP-110(Deltalzip) will also revert its effects upon actin filaments. Lastly, dominant-positive RhoA(V14) will block the ability of AFAP-110(Deltalzip) from inducing actin filament rosettes, but does not inhibit Src activation. Thus, conformational changes in AFAP-110 enable it to activate cellular kinases in a mechanism requiring SH3 and/or PH domain interactions. We hypothesize that cellular signals which alter AFAP-110 conformation, enable it to activate cellular kinases such as cSrc, which then direct changes in actin filament integrity in a Rho-dependent fashion.

Cr (VI) increases tyrosine phosphorylation through reactive oxygen species-mediated reactions.

While Cr (VI)-containing compounds are well established carcinogens, the mechanisms of their action remain to be investigated. In this study we show that Cr (VI) causes increased tyrosine phosphorylation in human lung epithelial A549 cells in a time-dependent manner. N-acetyl-cysteine (NAC), a general antioxidant, inhibited Cr (VI)-induced tyrosine phosphorylation. Catalase, a scavenger of H2O2, sodium formate and aspirin, scavengers of hydroxyl radical (*OH), also inhibited the increased tyrosine phosphorylation induced by Cr (VI). SOD, an inhibitor of superoxide radical (O2*-), caused less inhibition. ESR study shows that incubation of Cr (VI) with the A549 cells generates *OH radical. The generation of radical was decreased by addition of catalase and sodium formate, while SOD did not have any inhibitory effect. Oxygen consumption measurements show that addition of Cr (VI) to A549 cells resulted in enhanced molecular oxygen consumption. These results indicate that Cr (VI) can induce an increase in tyrosine phosphorylation. H2O2 and *OH radicals generated during the process are responsible for the increased tyrosine phosphorylation induced by Cr (VI).

The actin filament-associated protein AFAP-110 is an adaptor protein that modulates changes in actin filament integrity.

The actin filament-associated protein of 110 kDa (AFAP-110) was first identified as an SH3/SH2 binding partner for the nonreceptor tyrosine kinase, Src. Subsequent data have demonstrated that AFAP-110 can interact with other Src family members. AFAP-110 contains additional protein binding modules including two pleckstrin homology domains, a leucine zipper motif and a target sequence for serine/threonine phosphorylation. AFAP-110 interacts with actin filaments directly via a carboxy terminal actin-binding domain. Thus AFAP-110 may function as an adaptor protein by linking Src family members and/or other signaling proteins to actin filaments. AFAP-110 also has an intrinsic capability to alter actin filament integrity that can be revealed upon conformational changes associated with phosphorylation or mutagenesis. Recent data has indicated that AFAP-110 may also serve to activate cSrc in response to this conformational change as well. Thus, AFAP-110 may function in several ways by (1) acting as an adaptor protein that links signaling molecules to actin filaments, (2) serving as a platform for the construction of larger signaling complexes, (3) serving as an activator of Src family kinases in response to cellular signals that alter its conformation and (4) directly effecting actin filament organization as an actin filament cross-linking protein. Here, we will review the structure and function of AFAP-110 as well as potential binding partners and effectors of AFAP-110's ability to alter actin filament integrity.

Characterization of an alternatively spliced AATYK mRNA: expression pattern of AATYK in the brain and neuronal cells.

The AATYK gene encodes a tyrosine kinase whose expression is up-regulated during the apoptosis and differentiation of 32Dcl3 myeloblastic cells. Because high levels of AATYK mRNA have also been detected in the brain, and because these transcripts differ in size from that observed in the 32Dcl3 cell line, it was of interest to determine whether this gene encodes mRNAs that are alternatively spliced and whether these mRNAs are expressed in a tissue-specific manner. We have isolated a novel, alternatively spliced AATYK mRNA using cDNA library screening and RT-PCR, whose expression is readily detected in the brain but not myeloid cells. Western blot analysis revealed that the AATYK protein was expressed in virtually all regions of the adult rat brain in which neurons are present, including olfactory bulb, forebrain, cortex, midbrain, cerebellum and pons. Immunohistochemical labeling of adult brain sections showed the highest levels of AATYK expression in the cerebellum and olfactory bulb. Expression of AATYK was also up-regulated as a function of RA-induced neuronal differentiation of p19 embryonal carcinoma cells, supporting a role for this protein in mature neurons and neuronal differentiation.

Chimeric constructs containing the SH4/Unique domains of cYes can restrict the ability of Src(527F) to upregulate heme oxygenase-1 expression efficiently.

cSrc and cYes are the two most homologous members of the Src-family of nonreceptor tyrosine kinases. These kinases perform redundant signalling functions in cells; however, there is also evidence to support specificity in signalling. In this report, specificity in signalling between activated forms of the cSrc and cYes oncoproteins was examined at the level of downstream gene expression. Here, pp60c-src(527F) (Src(527F)) and chimeric constructs of Src(527F) containing combinations of the SH4/Unique/SH3/SH2 domains of cYes were generated to determine whether the individual modular domains of cSrc or cYes could direct distinct cellular signals leading to differential gene expression. A biased, differential display analysis approach was used to analyse changes in gene expression. The data indicate that Src(527F) is capable of upregulating heme oxygenase-1 (HO-1) in CEF cells at the level of transcription and protein expression. Chimeric constructs containing the SH4/Unique domains of cYes were less efficient in upregulating HO-1 expression. Activation of cSrc and expression of the HO-1 gene product are each induced under conditions of hypoxia. We hypothesize that activated cSrc can direct upregulation of HO-1 while activated cYes may be less efficient in stimulating signal transduction pathways that direct expression of HO-1.

Effects of the protein tyrosine phosphatase CD45 on FcgammaRIIa signaling and neutrophil function.

OBJECTIVE: Neutrophil receptors for the Fc portion of IgG (FcgammaR) trigger immune responses following cross-linking by IgG-coated foreign particles or immune complexes. Membrane-associated CD45, a protein tyrosine phosphatase termed leukocyte common antigen, has been shown to be essential for antigen receptor kinase mediated signaling in lymphocytes, and we hypothesized that CD45 may play a similar role in FcgammaR-mediated signaling and immune function in human neutrophils. METHODS: The experimental approach was that of cell surface molecule ligation via cross-linking with specific antibodies. Antibody dependent cellular cytotoxicity (ADCC) was assessed using a single-cell plaque assay and IL-6 production measured using ELISA. Tyrosine phosphorylation levels were assessed with anti-phospho-tyrosine blots and F-actin polymerization by flow cytometry and confocal microscopy. RESULTS: Neutrophils pretreated with anti-CD45 had a reduced ability to perform ADCC compared to untreated neutrophils. FcgammaRIIa cross-linking resulted in significantly increased concentrations of secreted IL-6 compared to untreated neutrophils, and IL-6 production was further enhanced by cocross-linking CD45 with FcgammaRIIa. Cross-linking CD45 alone also induced IL-6 production. FcgammaRIIa cross-linking resulted in increased protein tyrosine phosphorylation and F-actin polymerization in neutrophils. Cocross-linking CD45 with FcgammaRIIa resulted in abrogation of FcgammaRIIa mediated tyrosine phosphorylation and F-actin polymerization. CONCLUSIONS: These data provide evidence that CD45 can regulate or enhance the stimulation and function of human neutrophils mediated through FcgammaR(s). In addition, CD45 ligation may play an essential role in cytokine induction pathways that lead to inflammatory reactions in vivo.

The carboxy terminus of AFAP-110 modulates direct interactions with actin filaments and regulates its ability to alter actin filament integrity and induce lamellipodia formation.

The actin filament-associated protein AFAP-110 is an SH2/SH3 binding partner for Src. AFAP-110 contains several protein-binding motifs in its amino terminus and has been hypothesized to function as an adaptor molecule that could link signaling proteins to actin filaments. Recent studies using deletional mutagenesis demonstrated that AFAP-110 can alter actin filament integrity in SV40 transformed Cos-1 cells. Thus, AFAP-110 may be positioned to modulate the effects of Src upon actin filaments. In this report, we sought to determine whether (a) AFAP-110 could interact with actin filaments directly and (b) deletion mutants could affect actin filament integrity and cell shape in untransformed fibroblast cells. The data demonstrate that the carboxy terminus of AFAP-110 is both necessary and sufficient for actin filament association, in vivo and in vitro. Analysis of the carboxy terminus revealed a mean 40% similarity with other known actin-binding motifs, indicating a mechanism for binding to actin filaments. AFAP-110 can also induce lamellipodia formation. Contiguous with the alpha-helical, actin-binding motif is an alpha-helical, leucine zipper motif. Deletion of the leucine zipper motif (AFAP(Deltalzip)) followed by cellular expression enabled AFAP(Deltalzip) to alter actin filament integrity and cell shape in untransformed cells as evidenced by the induction of lamellipodia formation. We hypothesize that AFAP-110 may be an important signaling protein that can directly modulate changes in actin filament integrity and induce lamellipodia formation.

The SH3 and SH2 domains are capable of directing specificity in protein interactions between the non-receptor tyrosine kinases cSrc and cYes.

The c-src and c-yes proto-oncogenes encode 60 000 and 62 000 Dalton non-receptor tyrosine kinases of the Src family, pp60c-src and pp62c-yes, respectively. These kinases are over 80% homologous outside of their unique amino termini, yet several studies suggest that differences exist in the regulation, activation, and function of cSrc and cYes. The determinants of specificity in signaling between these proteins, however, remain unclear. In order to investigate the roles of the Src Homology (SH) 3 and 2 domains in mediating signaling specificity between cSrc and cYes, chimeras were created in which the SH3 and/or SH2 domains of cSrc or the fully activated variant Src527F were replaced by the corresponding domains of cYes. These constructs were used to assess the effects of the Yes SH3 and SH2 domains on the ability of Src to form stable complexes with and induce tyrosine phosphorylation of Src SH3 and SH2 domain binding partners in vivo. Both the Yes SH3 and SH2 domains were found to alter the capacity of Src to form stable associations with heterologous proteins. The Yes SH3 domain was unable to affinity absorb the Src SH3/SH2 binding partner AFAP-110 from COS-1 cell lysates, and chimeric constructs of Src527F containing the cYes SH3 domain were unable to efficiently co-immunoprecipitate with AFAP-110 from chicken embryo fibroblasts. Interactions with the Src SH2 domain binding partner pp130cas were unaffected. Additionally, only chimeras containing the cYes SH2 domain were able to co-immunoprecipitate with an unidentified 87 kDa tyrosine-phosphorylated protein. These results indicate that the SH3 and SH2 domains are capable of directing specificity in substrate binding between Src and Yes, suggesting potential mechanisms for generating specificity in signaling between these two highly related non-receptor tyrosine kinases.

Monoclonal antibodies directed against AFAP-110 recognize species-specific and conserved epitopes.

The actin filament-associated protein, AFAP-110, is a Src SH2/SH3 binding partner that can modulate changes in actin filament structure. AFAP-110 contains a carboxy terminal motif that facilitates actin filament interactions, as well as amino terminal protein binding motifs, including an SH3 binding motif, two SH2 binding motifs, and two Pleckstrin homology domains. Two monoclonal antibodies (MAbs) were developed that recognized epitopes in either the amino terminus (MAb 4C3) or the carboxy terminus (anti-AFAP-110) of AFAP-110. Site-directed mutations that change key proline residues to alanine in the SH3 binding motif and an adjacent proline-rich motif abrogated MAb 4C3 binding. These same mutations have been shown to prevent SH3 interactions between AFAP-110 and Src527F. These data indicate that MAb 4C3 recognizes an epitope that is part of the SH3 binding motif. Interestingly, MAb 4C3 is not efficiently reactive with mammalian homologs of AFAP-110. Sequence analysis of a putative cDNA clone that encodes the amino terminus of the human AFAP-110 isoform predicted a one amino acid difference within this epitope, indicating a mechanism for species-specific binding by MAb 4C3. A second, MAb anti-AFAP-110, recognizes AFAP-110 across species and binds to an epitope within the carboxy terminus. This epitope includes the 5th heptad repeat of the carboxy terminal, leucine zipper motif (amino acids 592-598)--a motif that facilitates self-associations and may regulate the function of AFAP-110. These MAbs will be useful for analyzing the effects of AFAP-110 upon cell morphology and actin filament integrity. In addition, the avian-specific MAb 4C3 may be useful for studying the effects of avian AFAP-110 constructs expressed in mammalian cells, by providing an internal epitope tag.

Formation of a stable src-AFAP-110 complex through either an amino-terminal or a carboxy-terminal SH2-binding motif.

The actin-filament-associated protein (AFAP-1 10) forms a stable complex with activated variants of the Pp60c-src (Src) non-receptor tyrosine kinase through SH2 and SH3 interactions. In this report, site-directed mutagenesis and a transient expression system that permits co-expression of activated pp60c-src (Src527F) and AFAP-110 in Cos-1 cells were used to identify the SH2-binding motif in AFAP-110. Four tyrosine residues, two in the amino terminus (Y93 and Y94) and two in the carboxy terminus (Y451 and Y453), were mutated to phenylalanine, significantly reducing overall steady-state levels of tyrosine phosphorylation and preventing Src527F from forming a stable complex with AFAP-110. These data indicate that the major sites for tyrosine phosphorylation are among these four tyrosine residues and that one or more of these tyrosines may function as an SH2-binding motif. Mutagenesis of just two tyrosines in either the amino terminus (Y93/Y94) or in the carboxy terminus (Y451/Y453) to phenylalanine had only a modest effect on steady-state levels of tyrosine phosphorylation and was not sufficient to abrogate stable-complex formation. These data suggest that Src527F can form a stable complex with AFAP-110 through either of two independently functional SH2-binding motifs. Triple-tyrosine mutation demonstrated that Y93 was not significantly phosphorylated on tyrosine and would not facilitate stable complex formation, whereas Y94, Y451, and Y453 could be phosphorylated on tyrosine and would facilitate stable-complex formation. We hypothesize that Src527F and AFAP-110 interact through a multistep binding mechanism that may either extend interactions between Src527F and actin filaments or permit reorientation of Src527F on AFAP-110, which could facilitate the presentation of Src527F toward other signaling molecules.

Src can regulate carboxy terminal interactions with AFAP-110, which influence self-association, cell localization and actin filament integrity.

The SH2 and SH3 binding partner AFAP-110 is a tyrosine phosphorylated substrate of Src. AFAP-110 has been hypothesized to link Src to actin filaments, which may contribute to the effects of Src upon actin filament integrity. However, it has been unclear what effect activated Src (Src527F) has upon AFAP-110 structure or function and whether AFAP-110 plays a role in actin filament integrity. We report here that the carboxy terminal 127 amino acids of AFAP-110 are comprised of an alpha-helical region that contains a leucine zipper motif. This indicated the potential of AFAP-110 to self-associate. Expression of the carboxy terminus as a fusion protein (GST-cterm) will permit affinity absorption of cellular AFAP-110. The integrity of the alpha-helical leucine zipper motif in GST-cterm is required for affinity absorption, but binding is not due to a classical leucine zipper interaction. Co-expression of Src527F, unlike cSrc, will abrogate affinity absorption of AFAP-110 with GST-cterm. These data indicate that Src527F has affected a change in the carboxy terminal structure that renders AFAP-110 unavailable for affinity absorption. Superose chromatography demonstrate that AFAP-110 will fractionate as a monomer or multimer, indicating AFAP-110 can be detected in a self-associated form in cell lysates. Co-expression of Src527F resulted in AFAP-110 fractionating with a molecular weight that predicts only a multimeric population. Deletional mutagenesis also indicate a biological role for the carboxy terminus in cellular localization and actin filament integrity. Deletion of the entire carboxy terminal alpha-helix (84 amino acids) will not permit AFAP-110 to efficiently colocalize with actin filaments or the cell membrane. Deletion of only the leucine zipper region of the carboxy terminal alpha-helix (44 amino acids) from AFAP-110 (AFAPAdeltazip) demonstrate that both AFAPdeltalzip and actin filaments are repositioned into rosette-like structures, similar to the effects of Src527F, while co-expression of AFAP-110 with cSrc will not affect actin filaments. These data indicate that AFAP-110 can play an important role in modulating actin filament integrity through carboxy terminal interactions that can be affected by Src527F.

The integrity of the SH3 binding motif of AFAP-110 is required to facilitate tyrosine phosphorylation by, and stable complex formation with, Src.

The actin filament-associated protein AFAP-110 forms a stable complex with activated variants of Src in chick embryo fibroblast cells. Stable complex formation requires the integrity of the Src SH2 and SH3 domains. In addition, AFAP-110 encodes two adjacent SH3 binding motifs and six candidate SH2 binding motifs. These data indicate that both SH2 and SH3 domains may work cooperatively to facilitate Src/AFAP-110 stable complex formation. As a test for this hypothesis, we sought to understand whether one or both SH3 binding motifs in AFAP-110 modulate interactions with the Src SH3 domain and if this interaction was required to present AFAP-110 for tyrosine phosphorylation by, and stable complex formation with, Src. A proline to alanine site-directed mutation in the amino terminal SH3 binding motif (SH3bm I) was sufficient to abrogate absorption of AFAP-110 with GST-SH3STC. Co-expression of activated Src (pp60(527F)) with AFAP-110 in Cos-1 cells permit tyrosine phosphorylation of AFAP-110 and stable complex formation with pp60(527F). However, co-expression of the SH3 null-binding mutant (AFAP71A) with pp60(527F) revealed a 2.7 fold decrease in steady-state levels of tyrosine phosphorylation, compared to AFAP-110. Although a lower but detectable level of AFAP71A was phosphorylated on tyrosine, AFAP71A could not be detected in stable complex with pp60(527F), unlike AFAP-110. These data indicate that SH3 interactions facilitate presentation of AFAP-110 for tyrosine phosphorylation and are also required for stable complex formation with pp60(527F).

Efficacy of taurine based compounds as hydroxyl radical scavengers in silica induced peroxidation.

While it is widely believed that taurine may play an important role in protecting cells against toxic injury by functioning as an antioxidant, there is a lack of evidence to support this hypothesis. In this study, electron spin resonance (ESR) was used to investigate the reaction of taurine and hypotaurine with hydroxyl radicals (.OH). The Fenton reaction (Fe(II) + H2O2-->Fe(III) + .OH + OH-) and the Cr(V)-mediated Fenton-like reaction (Cr(V) + H2O2-->Cr(VI) + .OH + OH-) were used as sources of .OH radicals. The results show that hypotaurine but not taurine effectively scavenges .OH radicals with a reaction rate constant of k = 1.6 x 10(10) M-1s-1. That is comparable with other efficient .OH radical scavengers. The effect of taurine and hypotaurine on silica-induced lipid peroxidation was evaluated using linoleic acid as a model lipid. Hypotaurine, but not taurine, caused a significant inhibition of silica-induced lipid peroxidation. The results show that hypotaurine is an excellent antioxidant and appears to have the potential for being a therapeutic agent against silica-induced lung injury.

Vanadium (IV) formation in the reduction of vanadate by glutathione reductase/NADPH and the role of molecular oxygen.

Experimental evidence documenting the formation of a relatively stable V(IV) species appears to be important with regard to the biochemical mechanism of reduction of vanadate by enzymatic systems. The present study demonstrates that a mixture of vanadate and glutathione reductase/nicotinamide-adenine-dinucleotide phosphate (NADPH), in phosphate (pH 7.2) buffer generates V(IV) under ambient conditions. Once formed, V(IV) does not rapidly autoxidize so as to defy detection by electron spin resonance (ESR) spectroscopy. The aerobic environment was guaranteed by preparing reaction mixtures in well stirred, wide mouth, standard test tubes in air over a period of 50 minutes, and by making ESR measurements in nuclear magnetic resonance (NMR) sample tubes as well as oxygen-permeable Teflon tubes. The V(IV) ESR signal intensity was found to increase linearly with time elapsed after reaction initiation. The linear growth of the V(IV) species also shows that this species is fairly stable, over a period of at least 50 minutes. Similar V(IV) stability data were obtained from VOSO4, a model compound as a source of V(IV). The results obtained in the present study demonstrated that V(IV) can be generated in the reduction of V(IV) by glutathione reductase in the presence of NADPH under aerobic condition.