Monilola A. Olayioye

The ErbB signaling network: receptor heterodimerization in development and cancer

Cells are continuously exposed to diverse stimuli ranging from soluble endocrine and paracrine factors, to signaling molecules on neighboring cells. It is of great importance that these extracellular signals are correctly interpreted by the cell, in order to achieve an appropriate developmental or proliferative response. Receptors of the tyrosine kinase family play pivotal roles in this process. By binding to specific peptide ligands they are able to integrate these external stimuli with internal signal transduction pathways, contributing in this fashion to the ability of the cell to respond correctly to its environment. In this review, we will concentrate on the role of ErbB receptors as normal signal transducers and their contribution to the process of malignant transformation during tumor development. ErbB proteins belong to subclass I of the superfamily of receptor tyrosine kinases (RTKs). There are four members of the ErbB family: epidermal growth factor (EGF) receptor (also termed ErbB1/HER1), ErbB2/Neu/HER2, ErbB3/HER3 and ErbB4/HER4. We will refer to them, henceforth, as the ErbB receptors. All family members have in common an extracellular ligand‐binding domain, a single membrane‐spanning region and a cytoplasmic protein tyrosine kinase domain. A family of ligands, the EGF‐related peptide growth factors, bind the extracellular domain of ErbB receptors leading to the formation of both homo‐ and heterodimers. Dimerization consequently stimulates the intrinsic tyrosine kinase activity of the receptors and triggers autophosphorylation of specific tyrosine residues within the cytoplasmic domain. These phosphorylated residues serve as docking sites for signaling molecules involved in the regulation of intracellular signaling cascades. Ultimately, downstream effects on gene expression determine the biological response to receptor activation. ErbB receptors are expressed in a variety of tissues of epithelial, mesenchymal and neuronal origin, where they play fundamental roles in development, proliferation and differentiation. Moreover, deregulated expression of ErbB receptors, in particular ErbB1 and ErbB2, has …

ErbB Receptor-induced Activation of Stat Transcription Factors Is Mediated by Src Tyrosine Kinases

Epidermal growth factor (EGF) binding to its receptor, ErbB1, triggers various signal transduction pathways, one of which leads to the activation of signal transducer and activator of transcription (Stat) factors. The mechanism underlying ErbB1-induced Stat activation and whether Stats are downstream targets of other ErbB receptors have not been explored. In this report we show that ErbB2, ErbB3, and ErbB4 do not potentiate Stat5 phosphorylation by EGF. However, neu differentiation factor-induced heterodimers of ErbB2 and ErbB4 activated Stat5. In A431 cells, Stat1, Stat3, and Stat5, were constitutively complexed with ErbB1 and rapidly phosphorylated on tyrosine in response to EGF. Neither mutation of the conserved tyrosine residue (Tyr694) nor inactivation of the Stat5a SH2 domain disrupted this association. However, an intact SH2 domain was necessary for EGF-induced Stat5a phosphorylation. In contrast to prolactin, which induced only Tyr694 phosphorylation of Stat5a, EGF promoted phosphorylation on Tyr694 and additional tyrosine residue(s). Janus kinases (Jaks) were also constitutively associated with ErbB receptors and were phosphorylated in response to EGF-related ligands. However, we provide evidence that EGF- and neu differentiation factor-induced Stat activation are dependent on Src but not Jak kinases. Upon EGF stimulation, c-Src was rapidly recruited to Stat/ErbB receptor complexes. Pharmacological Src kinase inhibitors and a dominant negative c-Src ablated both Stat and Jak tyrosine phosphorylation. However, dominant negative Jaks did not affect EGF-induced Stat phosphorylation. Taken together, the experiments establish two independent roles for Src kinases: (i) key molecules in ErbB receptor-mediated Stat signaling and (ii) potential upstream regulators of Jak kinases.

Intracellular signaling pathways of ErbB2/HER-2 and family members

ErbB (also termed HER) receptors are expressed in various tissues of epithelial, mesenchymal and neuronal origin, in which they are involved in the control of diverse biological processes such as proliferation, differentiation, migration and apoptosis. Furthermore, their deregulated expression has been implicated in many types of human cancers and is associated with poor clinical prognosis. Owing to the importance of ErbB proteins in both development and cellular transformation, a lot of attention has been drawn to the intracellular signals initiated by the engagement of this family of receptor tyrosine kinases. This review will focus on the membrane proximal events triggered by the ErbB receptor network and will address questions of how receptor heterodimerization may contribute to signal specification and diversification.

NDF/heregulin-induced cell cycle changes and apoptosis in breast tumour cells : role of PI3 kinase and p38 MAP kinase pathways

Neu differentiation factor (NDF)/heregulin activates ErbB2 via heterodimerization with the NDF receptors ErbB3 and ErbB4. Cells which express normal levels of these receptors are often growth stimulated by NDF, whereas SKBR3, and other ErbB2-overexpressing breast tumour cells are growth inhibited. We demonstrate here that in SKBR3 cells, NDF induces G1 progression but also causes a G2 delay from day 1 and apoptosis from days 2 – 3. G1 progression was associated with ErbB2 transactivation of ErbB3 and subsequent stimulation of the phosphatidylinositol 3-kinase (PI3K) pathway whereas apoptosis was dependent on p38 MAPK. Inhibition of ERK1/ERK2 had no effect on cell cycle progression or apoptosis. Activation of ErbB3 and PI3K was also seen with betacellulin (BTC) but not epidermal growth factor (EGF) and correlated with the growth effects of these ligands. All three ligands induced short-term activation of p38 MAPK in a c-Src-dependent manner. However, only NDF caused a second, c-Src-independent increase in p38 MAPK activity which was required for apoptosis.

Protein Kinase D Regulates Cell Migration by Direct Phosphorylation of the Cofilin Phosphatase Slingshot 1 Like

Protein kinase D (PKD) has been identified as a negative regulator of epithelial cell migration; however, its molecular substrates and downstream signaling pathways that mediate this activity have remained elusive. In this study, we provide evidence that the cofilin phosphatase slingshot 1 like (SSH1L), an important regulator of the complex actin remodeling machinery, is a novel in vivo PKD substrate. PKD-mediated phosphorylation of serines 937 and 978 regulates SSH1L subcellular localization by binding of 14-3-3 proteins and thus impacts the control of local cofilin activation and actin remodeling during cell migration. In line with this, we show that the loss of PKD decreases cofilin phosphorylation, induces a more spread cell morphology, and stimulates chemotactic migration of breast cancer cells in an SSHL1-dependent fashion. Our data thus identify PKD as a central regulator of the cofilin signaling network via direct phosphorylation and regulation of SSH1L.

DLC1 interacts with 14-3-3 proteins to inhibit RhoGAP activity and block nucleocytoplasmic shuttling.

Deleted in liver cancer 1 (DLC1) is a Rho-GTPase-activating protein (GAP) that is downregulated in various tumor types. In vitro, DLC1 specifically inactivates the small GTPases RhoA, RhoB and RhoC through its GAP domain and this appears to contribute to its tumor suppressor function in vivo. Molecular mechanisms that control DLC1 activity have not so far been investigated. Here, we show that phorbol-ester-induced activation of protein kinase C and protein kinase D stimulates association of DLC1 with the phosphoserine/phosphothreonine-binding 14-3-3 adaptor proteins via recognition motifs that involve Ser327 and Ser431. Association with 14-3-3 proteins inhibits DLC1 GAP activity and facilitates signaling by active Rho. We further show that treatment of cells with phorbol ester or coexpression of 14-3-3 proteins, blocks DLC1 nucleocytoplasmic shuttling, probably by masking a previously unrecognized nuclear localization sequence. The binding to 14-3-3 proteins is thus a newly discovered mechanism by which DLC1 activity is regulated and compartmentalized.

Tenascin-C blocks cell-cycle progression of anchorage-dependent fibroblasts on fibronectin through inhibition of syndecan-4

Tenascin-C is an adhesion-modulatory extracellular matrix protein that is predominantly expressed during embryonic development, wound healing and in tumor stroma. Here we report that anchorage-dependent human, rat and mouse fibroblasts adhere poorly and fail to proliferate on pure tenascin-C. This was due to a significant reduction of cyclin-dependent kinase 2 (cdk2) activity, resulting from elevated expression and association of the cdk inhibitors (CKIs) p21Cip1 and p27Kip1. To analyse the effect of tenascin-C on fibronectin-mediated adhesion, cells were plated on a mixed fibronectin/tenascin-C substratum. Compared to fibronectin alone, cell spreading and adhesion signaling were compromised, as determined by delayed phosphorylation kinetics of focal adhesion kinase (FAK). Despite the presence of growth factors, these cells remained arrested in the G1 phase of the cell cycle. In contrast to cells plated on pure tenascin-C, cdk2 activity appeared to be inhibited independently of CKIs. Interestingly, overexpression of the transmembrane proteoglycan syndecan-4 restored cell spreading, adhesion signaling and DNA replication on the fibronectin/tenascin-C substratum. A similar rescue was observed using a recombinant peptide that spans the syndecan-4-binding site in fibronectin. This indicates that tenascin-C causes cell cycle arrest and cdk2 inactivation by interfering with fibronectin–syndecan-4 interactions. We therefore propose that syndecan-4 signaling plays a central role in the control of cellular proliferation of anchorage-dependent fibroblasts.

XIAP-deficiency leads to delayed lobuloalveolar development in the mammary gland

Inhibitors of apoptosis proteins (IAPs) were originally identified in baculoviruses where they prevent apoptosis of the host cell, thereby allowing viral propagation. Cellular homologues of the viral IAP have been identified in yeast and metazoans. IAPs share a conserved structure known as the baculovirus IAP repeat (BIR) domain, which is an B80 amino acid zincfinger motif. These proteins include DIAP1 and DIAP2 in Drosophila, XIAP, cIAP1, cIAP2 and ML-IAP as well as the more distantly related family members NAIPs, Survivin and BRUCE in vertebrates. Some IAP proteins can act as potent inhibitors of apoptosis and appear to be the only endogenous inhibitors that directly inhibit both initiator and effector caspases. The most extensively studied IAP is X-linked inhibitor of apoptosis protein (XIAP). This protein comprises three BIR domains at its N-terminus and a RING domain that has E3 ubiquitin ligase activity at its C-terminus. Cells transfected with XIAP are protected from apoptosis in response to a range of stimuli. XIAP directly binds to and inhibits caspases 3, 7 and 9. Although XIAP is ubiquitously expressed, XIAP-deficient mice were not reported to have any gross phenotypic abnormalities or any profound defects in programmed cell death following the induction of apoptosis by a variety of stimuli. Recently, however, XIAP-deficient sympathetic neurons were reported to be more susceptible to cytochrome c-induced cell death. We have found that XIAP expression is developmentally regulated in the mammary gland (see below) and therefore investigated a potential role for this gene during mammopoiesis using targeted mice. Homologous recombination in embryonic stem (ES) cells was used to disrupt the murine XIAP locus, which comprises six coding exons (Figure 1a). The targeting vector was designed to replace exons 1 and 2 with a PGK-neomycin resistance cassette, thus deleting the initiation codon, the first two BIR domains and the majority of the third BIR domain (Figure 1a). Several clonal G418targeted ES cell lines were generated and correct integration was verified by Southern blot analysis using the indicated probe (Figure 1b). Chimeric mice generated from two independent ES cell lines (239 and 240) gave germ-line transmission and were used to generate heterozygous XIAP female mice. These were intercrossed to yield male and female mice lacking XIAP. To confirm that deletion of exons 1 and 2 within the XIAP locus gave rise to animals deficient in XIAP, we performed Western blot analysis using a monoclonal XIAP antibody. XIAP was not detectable in XIAP mutant tissues including the liver, kidney, spleen and mammary gland, whereas it was readily visualized in tissues from wild-type mice (Figure 1e and data not shown). Therefore, targeted disruption of the XIAP gene generated a null mutation. Consistent with previous findings, XIAP-deficient mice appeared healthy with no gross defects. XIAP expression was examined in the mouse mammary gland by Western blotting. The expression was found to vary during mammary gland development, with highest levels evident in late pregnancy (Figure 1c). We therefore examined whether mammary gland development was normal in XIAPnull mice. No defects were evident in the mammary glands of virgin (n1⁄4 3), lactating (n1⁄4 8) or involuting (n1⁄4 8) female mice. However, histological and wholemount analyses of mammary glands from two independent strains of XIAPdeficient mice at different stages of pregnancy revealed a defect during late pregnancy (Figure 1d). Reduced lobuloalveolar development was evident in the mammary glands of all XIAP / mice at day 16 (n1⁄4 3), day 17 (n1⁄4 3) and day 18 (n1⁄4 5) of pregnancy, compared with that seen in wild-type control animals. The density of lobuloalveoli was significantly decreased and the lumens were less dilated, suggesting reduced milk protein synthesis and/or secretion (Figure 1d). The delay in lobuloalveolar development was also evident at 0.5 day lactation but was no longer apparent by day 7 of lactation. Consistent with these observations, females were found to lactate normally. RT-PCR analysis of cIAP-1 and cIAP-2 expression revealed that mRNA levels were similar in mutant and wild-type mammary glands during late pregnancy and early lactation (data not shown). Therefore, compensatory upregulation of these IAP genes does not appear to occur in the mammary glands of XIAP-null mice, suggesting an alternative mechanism for the transient nature of the phenotype observed. Incorporation of BrdU was used to assess the number of proliferating cells at 16 and 18 days of pregnancy in XIAP / and wild-type mice. However, no marked alteration in the number of proliferating cells was evident between wild-type and mutant glands (data not shown). Nor was there any significant change in the number of apoptotic cells as detected by TUNEL staining (data not shown). These data indicate that proliferation and apoptosis are not substantially altered in cells in XIAP / mammary glands, suggesting that mammary differentiation may be aberrant in these mice. To determine whether the condensed acini observed in mammary glands of XIAP-deficient mice reflected a decrease Cell Death and Differentiation (2005) 12, 87–90 & 2005 Nature Publishing Group All rights reserved 1350-9047/05

Deleted in Liver Cancer 1 Controls Cell Migration through a Dia1-Dependent Signaling Pathway

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Heterologous expression of the lipid transfer protein CERT increases therapeutic protein productivity of mammalian cells.

Deleted in liver cancer (DLC) 1 and 2 are Rho GTPase-activating proteins that are frequently down-regulated in various types of cancer. Ectopic expression in carcinoma cell lines lacking these proteins has been shown to inhibit cell migration and invasion. However, whether the loss of DLC1 or DLC2 is the cause of aberrant Rho signaling in transformed cells has not been investigated. Here, we have down-regulated DLC1 and DLC2 expression in breast cancer cells using a RNA interference approach. Silencing of DLC1 led to the stabilization of stress fibers and focal adhesions and enhanced cell motility in wound-healing as well as chemotactic Transwell assays. We provide evidence that enhanced migration of cells lacking DLC1 is dependent on the Rho effector protein Dia1 but does not require the activity of Rho kinase. By contrast, DLC2 knockdown failed to affect the migratory behavior of cells, suggesting that the two proteins have distinct functions. This is most likely due to their differential subcellular localizations, with DLC1 found in focal adhesions and DLC2 being mainly cytosolic. Collectively, our data show that DLC1 is critically involved in the control of Rho signaling and actin cytoskeleton remodeling and that its cellular loss is sufficient for the acquisition of a more migratory phenotype of breast cancer cells.