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.

Interleukin-3 protects Bcr-Abl-transformed hematopoietic progenitor cells from apoptosis induced by Bcr-Abl tyrosine kinase inhibitors.

Bcr-Abl tyrosine kinase has been validated as a molecular target for the treatment of chronic myelogenous leukemia (CML). More recently, it has been reported that CML patients could develop resistance to the Bcr-Abl tyrosine kinase inhibitor, imatinib (STI571, Gleevec), pointing to the need for development of additional Bcr-Abl tyrosine kinase inhibitors or other therapeutic strategies. It was also found that a significant proportion of patients who received the Bcr-Abl inhibitor did not achieve complete cytogenetic response. Mechanisms for incomplete cytogenetic response to Bcr-Abl inhibition are not entirely clear. We report here three new pyrido[2,3-d]pyrimidine Bcr-Abl tyrosine kinase inhibitors, PD164199, PD173952, PD173958, that induced apoptosis of Bcr-Abl-dependent hematopoietic cells. An interleukin-3 (IL-3) autocrine loop was observed previously in primitive CD34(+)/Bcr-Abl(+) leukemic cells in CML patients. Using 32Dp210(Bcr-Abl)and Baf3p210(Bcr-Abl) cells as models, we tested whether IL-3 might protect Bcr-Abltransformed, IL-3-responsive cells from apoptosis caused by Bcr-Abl tyrosine kinase inhibition. Results of trypan blue exclusion, fluoroisothiocyanate-valyl-alanyl-aspartyl-[O-methyl] -fluoromethylketone (FITC-VAD-FMK), and Annexin-V/7-amino-actinomycin D (7-AAD) binding assays indicate that IL-3 could protect Bcr-Abl-transformed, IL-3 responsive hematopoietic progenitor cells from apoptosis induced by Bcr-Abl tyrosine kinase inhibitors. This finding raises the possibility that the IL-3 autocrine loop found in primitive CD34(+)/Bcr-Abl(+) cells in CML patients could contribute to the incomplete eradication of Bcr-Abl(+) cells by Bcr-Abl inhibition.

Phosphotyrosines 627 and 659 of Gab1 constitute a bisphosphoryl tyrosine-based activation motif (BTAM) conferring binding and activation of SHP2.

A major Grb2-associated binder-1 (Gab1) binding partner in epidermal growth factor (EGF)-stimulated cells is protein-tyrosine phosphatase (PTPase) SHP2, which contains tandem SH2 domains. The SHP2 PTPase activity is required for activation of the extracellular signal-regulated kinase (ERK) subfamily of mitogen-activated protein (MAP) kinase by EGF. To investigate the mechanism by which Gab1 and SHP2 mediate ERK activation, we characterized the Gab1-SHP2 interaction. We found that both Tyr-627 and Tyr-659 of Gab1 were required for SHP2 binding to Gab1 and for ERK2 activation by EGF. Far Western blot analysis suggested that the tandem SH2 domains of SHP2 bind to Gab1 in a specific orientation, in which the N-SH2 domain binds to phosphotyrosine (Tyr(P))-627 and the C-SH2 domain binds to Tyr(P)-659. When assayed with peptide substrates, SHP2 PTPase was activated by a bisphosphopeptide containing both Tyr(P)-627 and Tyr(P)-659, but not by monophosphopeptides containing Tyr(P)-627 or Tyr(P)-659 or a mixture of these monophosphopeptides. These results suggest that Tyr(P)-627 and Tyr(P)-659 of Gab1 constitute a bisphosphoryl tyrosine-based activation motif (BTAM) that binds and activates SHP2. Remarkably, while a constitutively active SHP2 (SHP2DeltaN) could not rescue the defect of a SHP2-binding defective Gab1 (Gab1FF) in ERK2 activation, expression of a Gab1FF-SHP2DeltaN chimera resulted in constitutive activation of ERK2 in transfected cells. Thus, physical association of activated SHP2 with Gab1 is necessary and sufficient to mediate the ERK mitogen-activated protein kinase activation. Phosphopeptides derived from Gab1 were dephosphorylated by active SHP2 in vitro. Consistently, substrate-trapping experiments with a SHP2 catalytic inactive mutant suggested that Gab1 was a SHP2 PTPase substrate in the cells. Therefore, Gab1 not only is a SHP2 activator but also is a target of its PTPase.

Requirement of SHP2 binding to Grb2-associated binder-1 for mitogen-activated protein kinase activation in response to lysophosphatidic acid and epidermal growth factor.

Grb2-associated binder-1 (Gab1) is a multisite docking protein containing a pleckstrin homology (PH) domain, multiple potential tyrosine phosphorylation sites, and several proline-rich sequences. Gab1 becomes tyrosine-phosphorylated in cells stimulated with growth factors, cytokines, and ligands for G protein-coupled receptors. A major Gab1-binding protein detected in cells treated with extracellular stimuli is the tyrosine phosphatase, SHP2. Although the role of SHP2-Gab1 interaction in cell signaling has not yet been characterized, SHP2 is known to mediate mitogen-activated protein (MAP) kinase activation induced by the epidermal growth factor (EGF). However, the mechanism by which the SHP2 phosphatase exerts a positive signaling role remains obscure. In this study, we prepared Gab1 mutants lacking the SHP2 binding site (Gab1Y627F), the phosphatidylinositol 3-kinase (PI3K) binding sites (Gab1DeltaPI3K), and the PH domain (Gab1DeltaPH). Expression of Gab1Y627F blocked the extracellular signal-regulated kinase-2 (ERK2) activation by lysophosphatidic acid (LPA) and EGF. Conversely, expression of the wild-type Gab1 in HEK293 cells augmented the LPA receptor Edg2-mediated ERK2 activation. Whereas the PH domain was required for Gab1 mediation of ERK2 activation by LPA, it was not essential for EGF-induced ERK2 activation. Expression of Gab1DeltaPI3K had no apparent effect on ERK2 activation by LPA and EGF in the cells that we have examined. These results establish a role for Gab1 in the LPA-induced MAP kinase pathway and clearly demonstrate that Gab1-SHP2 interaction is essential for ERK2 activation by LPA and EGF. These findings also suggest that the positive role of SHP2 in the MAP kinase pathway depends on its interaction with Gab1.

Involvement of lipoxygenase in lysophosphatidic acid-stimulated hydrogen peroxide release in human HaCaT keratinocytes.

Although it is now recognized that low levels of reactive oxygen species (ROS) are required for the mitogenic response, mitogen-induced signalling pathways that regulate ROS generation in non-phagocytic cells remain largely uncharacterized. Using a real-time assay for measuring hydrogen peroxide (H(2)O(2)) formation, we analysed H(2)O(2) release in human HaCaT keratinocytes in response to lysophosphatidic acid (LPA), a mitogen for keratinocytes. LPA rapidly increased H(2)O(2) release in HaCaT cells. Unlike LPA-induced mitogen-activated protein (MAP) kinase activation, LPA-stimulated H(2)O(2) release was independent of the tyrosine kinase activity of the epidermal growth factor (EGF) receptor. Calcium chelators, phospholipase A(2) inhibitors, and lipoxygenase inhibitors effectively blocked LPA-stimulated H(2)O(2) release, whereas cyclooxygenase inhibitors were without effect. Addition of 5-lipoxygenase products 5-hydroperoxyeicosatetraenoic acid (5-HPETE) and leukotriene B(4), but not 5-hydroxyeicosatetraenoic acid (5-HETE) and leukotriene C(4), restored LPA-stimulated H(2)O(2) release in cells treated with the lipoxygenase inhibitors nordihydroguaiaretic acid and Zileuton. These results suggest that the lipoxygenase products 5-HPETE and leukotriene B(4) are required for LPA-stimulated H(2)O(2) release in HaCaT cells.

Reversible regulation of SHP-1 tyrosine phosphatase activity by oxidation.

Increasing evidence indicates that redox regulation is an important signaling mechanism. Protein tyrosine phosphatases (PTPases) are sensitive to oxidative inactivation and are potential targets of redox regulation. In this study, we analyzed the reversibility of oxidative inactivation of the PTPase SHP-1, which negatively regulates protein tyrosine kinase signaling. H2O2 inactivated SHP-1 in vitro. Incubation of the H2O2-inactivated SHP-1 with dithiothreitol recovered 44-99% of the PTPase activity, depending on the H2O2 concentrations used to inactivate SHP-1. Glutathione and N-acetylcysteine also reactivated H2O2-treated SHP-1. Stimulation of SHP-1-transfected HeLa cells with H2O2 rapidly decreased SHP-1 activity, which was completely reversed within 15 min. Thus, oxidative inactivation of SHP-1 is a reversible process.

Role of tyrosine kinase activity of epidermal growth factor receptor in the lysophosphatidic acid-stimulated mitogen-activated protein kinase pathway.

Recent evidence indicates that the epidermal growth factor (EGF) receptor mediates a branch of lysophosphatidic acid (LPA)-induced signal transduction pathways that activate mitogen-activated protein (MAP) kinase. However, it is unclear whether the intrinsic tyrosine kinase activity of EGF receptor is involved. We previously showed that reactive oxygen species (ROS) were involved in the LPA-stimulated MAP kinase pathway. Here, we identify tyrosine phosphorylation of EGF receptor as an LPA signaling step that requires ROS. To evaluate the role of the tyrosine kinase activity of EGF receptor in the LPA-stimulated MAP kinase pathway, we examined the effects of an EGF receptor-specific tyrosine kinase inhibitor, PD158780. PD158780 potently inhibited the LPA-stimulated MAP kinase kinase 1/2 (MKK1/2) activation and EGF receptor tyrosine phosphorylation in HeLa cells, while it had no detectable effect on c-Src kinase activity. PD158780 also inhibited LPA-induced MKK1/2 activation and DNA synthesis in NIH 3T3 cells. Furthermore, we compared LPA-stimulated MKK1/2 and MAP kinase activation, transcriptional activity of the c-fos promoter, and DNA synthesis in B82L cells, which lack endogenous EGF receptor, and B82L cells expressing kinase-defective or wild-type human EGF receptor. Results obtained from analysis of these cell lines suggest that the EGF receptor tyrosine kinase contributes to the LPA-stimulated MAP kinase activation, c-fos transcription, and mitogenesis.

Suppression of fibroblast cell cycle progression in G1 phase by N-acetylcysteine.

The antioxidant N-acetyl-L-cysteine (NAC) has been increasingly used as an experimental tool to assess involvement of reactive oxygen species in cell signaling and is being evaluated as a preventive and therapeutic agent for cancer and pulmonary diseases related to inflammation and oxidative stress. However, a detailed characterization of the effect of NAC on cell cycle progression has not been reported. In the present study, modulation of cell cycle progression by NAC was analyzed in mouse fibroblast NIH3T3 cells grown in 10% fetal bovine serum. Complete inhibition of NIH3T3 cell proliferation was obtained with 20 mM NAC. Inhibition of cell proliferation by NAC (at or below 20 mM) was not due to cell death, and the antiproliferative effect of NAC was reversible. Flow cytometric analysis of cell cycle phase distribution indicated that NAC blocked the cell cycle in the G1 phase. Consistent with this observation, NAC inhibited DNA synthesis. After releasing the G1-block by NAC, S phase re-entry occurred between 8 and 12 h, suggesting that NAC blocked the cell cycle in early to mid-G1 phase. NAC prevented activation of mitogen-activated protein (MAP) kinases p42MAPK and p44MAPK and inhibited expression of cyclin D1, but had no effect on the levels of proliferating cell nuclear antigen. Incubation of cells with PD98059, a specific inhibitor of MAP kinase kinase 1, partially arrested the cell cycle in the G1 phase. These results indicate that the antiproliferative effect of NAC is linked in part to inhibition of the MAP kinase pathway.

Identification of the retinal cyclic GMP phosphodiesterase inhibitory gamma-subunit interaction sites on the catalytic alpha-subunit.

Retinal rod outer segment phosphodiesterase (PDE) consists of two similar catalytic subunits (alpha and beta) and two identical inhibitory subunits (gamma 2). A trypsin-activated soluble PDE exhibiting the ability to be reinhibited by PDE gamma was shown by peptide antisera to retain both N and C termini. Synthetic peptides corresponding to residues 16-30, 78-90, 389-403, and 535-563 of PDE alpha used in a PDE activity assay with trypsin-activated PDE partially prevented inhibition by exogenous PDE gamma; however, only competitions by peptides 16-30 and 78-90 (corresponding to PDE alpha 16-30 and 78-90) were concentration-dependent below 100 nmol of peptide. Binding studies using radio-immunoassays and PDE alpha peptides confirmed that peptides 16-30 and 78-90 (corresponding to PDE alpha 16-30 and 78-90, respectively) were able to bind PDE gamma. Additionally, peptides corresponding to the PDE alpha region 453-534 bound PDE gamma in the binding assay. This suggests that several regions on PDE alpha interact with the PDE gamma inhibitor. While some regions may be involved in binding to PDE gamma, other sites may be involved in PDE gamma inhibition of catalytic activity. Our results suggest that the major regions of PDE alpha that interact with PDE gamma reside within the N terminus (16-30 and 78-90), with weaker interaction regions within or near the hypothesized catalytic domain (453-563). Sequence analysis of three retinal phosphodiesterases (rod outer segment alpha, beta, and cone outer segment alpha') revealed the highest region of dissimilarity in the N and C termini.

Binding of the gamma-subunit of retinal rod-outer-segment phosphodiesterase with both transducin and the catalytic subunits of phosphodiesterase.

The gamma-subunit of retinal rod-outer-segment phosphodiesterase (PDE-gamma) is a multifunctional protein which interacts directly with both of the catalytic subunits of PDE (PDE alpha/beta) and the alpha-subunit of the retinal G (guanine-nucleotide-binding)-protein transducin alpha (T alpha). We have previously reported that the PDE gamma binds to T alpha at residue nos. 24-45 [Morrison. Rider & Takemoto (1987) FEBS Lett. 222, 266-270]. In vitro this results in inhibition of T alpha GTP/GDP exchange [Morrison, Cunnick, Oppert & Takemoto (1989) J. Biol. Chem. 264, 11671-11681]. We now report that the inhibitory region of PDE gamma for PDE alpha/beta occurs at PDE gamma residues 54-87. This binding results in inhibition of either trypsin-solubilized or membrane-bound PDE alpha/beta. PDE gamma which has been treated with carboxypeptidase Y, removing the C-terminus, does not inhibit PDE alpha/beta, but does inhibit T alpha GTP/GDP exchange. Inhibition by PDE gamma can be removed by T alpha-guanosine 5'-[gamma-thio]triphosphate (GTP[S]) addition to membranes. This results in a displacement of PDE gamma, but not in removal of this subunit from the membrane [Whalen, Bitensky & Takemoto (1990) Biochem. J. 265, 655-658]. These results suggest that low levels of T alpha-GTP[S] can result in displacement of PDE gamma from the membrane in vitro as a GTP[S]-T alpha-PDE gamma complex. Further activation by high levels of T alpha-GTP[S] occurs by displacement of PDE gamma from its inhibitory site on PDE alpha/beta, but not in removal from the membrane.

Visual transduction in rod outer segments.

The visual transduction cascade of the retinal rod outer segment responds to light by decreasing membrane current. This ion channel is controlled by cyclic GMP which is, in turn, controlled by its synthesis and degradation by guanylate cyclase and phosphodiesterase, respectively. When light bleaches rhodopsin there is an induced exchange of GTP for GDP bound to the alpha subunit of the retinal G-protein, transducin (T). The T alpha.GTP then removes the inhibitory constraint of a small inhibitory subunit (PDE gamma) on the retinal cGMP phosphodiesterase (PDE). This results in activation of the PDE and in hydrolysis of cGMP. Recently both low and high affinity binding sites have been identified for PDE gamma on the PDE alpha/beta catalytic subunits. The discovery of two PDE gamma subunits, each with different binding affinities, suggests that a tightly regulated shut-off mechanism may be present.

Interaction of the gamma-subunit of retinal rod outer segment phosphodiesterase with transducin. Use of synthetic peptides as functional probes.

There is considerable evidence which suggests that the gamma-subunit of cGMP phosphodiesterase (PDE gamma) is a multifunctional protein which may interact directly with both the catalytic subunits of PDE (PDE alpha beta) and the alpha-subunit of transducin (T alpha) (Whalen, M., and Bitensky, M. (1989) Biochem. J. 259, 13-19; Griswold-Prenner, I., Young, J. H., Yamane, H. K., and Fung, B. K.-K. (1988) Invest. Ophthalmol. & Visual Sci. 29, (Suppl.) 218). To determine the region of interaction between the multifunctional PDE gamma and T alpha, and to determine the significance of this interaction, peptides corresponding to various regions of PDE gamma were synthesized and tested for their ability to inhibit the GTPase activity of T alpha. One of these peptides, PDE gamma-3 (bovine amino acid residues 31-45), inhibited the GTPase activity of T alpha with an I50 of 450 microM. The peptide (PDE gamma-3) was found to inhibit the GTPase activity of T alpha by inducing the binding of transducin to the rod outer segment membrane and by altering the GTP/GDP exchange. Analogs of PDE gamma-3 were synthesized to determine the required structure of the PDE gamma-3 region needed for the interaction of PDE gamma with T alpha. The results of these studies indicated that the removal of the positively charged amino acids or any of the potential hydrogen-bonding amino acids increased the I50 for the inhibition of the GTPase activity of T alpha Substitution of the hydrophobic amino acids had no effect. These results indicate the hydrophilic interactions may be essential for the binding of PDE gamma to T alpha and for the inhibition of the GTPase activity of T alpha by PDE gamma. The observed effects of PDE gamma-3 on T alpha and on PDE suggest that PDE gamma is a multifunctional protein which may play more than one role in the deactivation of the retinal transduction cascade.