Grb2 monomer-dimer equilibrium determines normal versus oncogenic function

The adaptor protein growth factor receptor-bound protein 2 (Grb2) is ubiquitously expressed in eukaryotic cells and involved in a multitude of intracellular protein interactions. Grb2 plays a pivotal role in tyrosine kinase-mediated signal transduction including linking receptor tyrosine kinases to the Ras/mitogen-activated protein (MAP) kinase pathway, which is implicated in oncogenic outcome. Grb2 exists in a constitutive equilibrium between monomeric and dimeric states. Here we show that only monomeric Grb2 is capable of binding to SOS and upregulating MAP kinase signalling and that the dimeric state is inhibitory to this process. Phosphorylation of tyrosine 160 (Y160) on Grb2, or binding of a tyrosylphosphate-containing ligand to the SH2 domain of Grb2, results in dimer dissociation. Phosphorylation of Y160 on Grb2 is readily detectable in the malignant forms of human prostate, colon and breast cancers. The self-association/dissociation of Grb2 represents a switch that regulates MAP kinase activity and hence controls cancer progression

Reduced Estradiol-Induced Vasodilation and Poly-(ADP-Ribose) Polymerase (PARP) Activity in the Aortas of Rats with Experimental Polycystic Ovary Syndrome (PCOS)

Polycystic ovary syndrome (PCOS) is a complex endocrine disorder characterized by hyperandrogenism and insulin resistance, both of which have been connected to atherosclerosis. Indeed, an increased risk of clinical manifestations of arterial vascular diseases has been described in PCOS. On the other hand endothelial dysfunction can be detected early on, before atherosclerosis develops. Thus we assumed that vascular dysfunction is also related directly to the hormonal imbalance rather than to its metabolic consequences. To detect early functional changes, we applied a novel rodent model of PCOS: rats were either sham operated or hyperandrogenism was achieved by implanting subcutaneous pellets of dihydrotestosterone (DHT). After ten weeks, myograph measurements were performed on isolated aortic rings. Previously we described an increased contractility to norepinephrine (NE). Here we found a reduced immediate relaxation to estradiol treatment in pre-contracted aortic rings from hyperandrogenic rats. Although the administration of vitamin D3 along with DHT reduced responsiveness to NE, it did not restore relaxation to estradiol. Poly-(ADP-ribose) polymerase (PARP) activity was assessed by poly-ADP-ribose immunostaining. Increased PAR staining in ovaries and circulating leukocytes from DHT rats showed enhanced DNA damage, which was reduced by concomitant vitamin D3 treatment. Surprisingly, PAR staining was reduced in both the endothelium and vascular smooth muscle cells of the aorta rings from hyperandrogenic rats. Thus in the early phase of PCOS, vascular tone is already shifted towards vasoconstriction, characterized by reduced vasorelaxation and vascular dysfunction is concomitant with altered PARP activity. Based on our findings, PARP inhibitors might have a future perspective in restoring metabolic disorders in PCOS

The Tyrosine Kinase c-Src Directly Mediates Growth Factor-Induced Notch-1 and Furin Interaction and Notch-1 Activation in Pancreatic Cancer Cells

The proteolytic activity of Furin responsible for processing full length Notch-1 (p300) plays a critical role in Notch signaling. The amplitude and duration of Notch activity can be regulated at various points in the pathway, but there has been no report regarding regulation of the Notch-1-Furin interaction, despite its importance. In the present study, we found that the Notch-1-Furin interaction is regulated by the non-receptor tyrosine kinase, c-Src. c-Src and Notch-1 are physically associated, and this association is responsible for Notch-1 processing and activation. We also found that growth factor TGF-α, an EGFR ligand, and PDGF-BB, a PDGFR ligand, induce the Notch-1-Furin interaction mediated by c-Src. Our results support three new and provocative conclusions: (1) The association between Notch-1 and Furin is a well-regulated process; (2) Extracellular growth factor signals regulate this interaction, which is mediated by c-Src; (3) There is cross-talk between the plasma growth factor receptor-c-Src and Notch pathways. Co-localization of Notch-1 and c-Src was confirmed in xenograft tumor tissues and in the tissues of pancreatic cancer patients. Our findings have implications for the mechanism by which the Notch and growth factor receptor-c-Src signaling pathways regulate carcinogenesis and cancer cell growth

Intrinsic Structural Disorder Confers Cellular Viability on Oncogenic Fusion Proteins

Chromosomal translocations, which often generate chimeric proteins by fusing segments of two distinct genes, represent the single major genetic aberration leading to cancer. We suggest that the unifying theme of these events is a high level of intrinsic structural disorder, enabling fusion proteins to evade cellular surveillance mechanisms that eliminate misfolded proteins. Predictions in 406 translocation-related human proteins show that they are significantly enriched in disorder (43.3% vs. 20.7% in all human proteins), they have fewer Pfam domains, and their translocation breakpoints tend to avoid domain splitting. The vicinity of the breakpoint is significantly more disordered than the rest of these already highly disordered fusion proteins. In the unlikely event of domain splitting in fusion it usually spares much of the domain or splits at locations where the newly exposed hydrophobic surface area approximates that of an intact domain. The mechanisms of action of fusion proteins suggest that in most cases their structural disorder is also essential to the acquired oncogenic function, enabling the long-range structural communication of remote binding and/or catalytic elements. In this respect, there are three major mechanisms that contribute to generating an oncogenic signal: (i) a phosphorylation site and a tyrosine-kinase domain are fused, and structural disorder of the intervening region enables intramolecular phosphorylation (e.g., BCR-ABL); (ii) a dimerisation domain fuses with a tyrosine kinase domain and disorder enables the two subunits within the homodimer to engage in permanent intermolecular phosphorylations (e.g., TFG-ALK); (iii) the fusion of a DNA-binding element to a transactivator domain results in an aberrant transcription factor that causes severe misregulation of transcription (e.g. EWS-ATF). Our findings also suggest novel strategies of intervention against the ensuing neoplastic transformations

The transcriptional activity of hepatocyte nuclear factor 4 alpha is inhibited via phosphorylation by ERK1/2

Hepatocyte nuclear factor 4 alpha (HNF4alpha) nuclear receptor is a master regulator of hepatocyte development, nutrient transport and metabolism. HNF4alpha is regulated both at the transcriptional and post-transcriptional levels by different mechanisms. Several kinases (PKA, PKC, AMPK) were shown to phosphorylate and decrease the activity of HNF4alpha. Activation of the ERK1/2 signalling pathway, inducing proliferation and survival, inhibits the expression of HNF4alpha. However, based on our previous results we hypothesized that HNF4alpha is also regulated at the post-transcriptional level by ERK1/2. Here we show that ERK1/2 is capable of directly phosphorylating HNF4alpha in vitro at several phosphorylation sites including residues previously shown to be targeted by other kinases, as well. Furthermore, we also demonstrate that phosphorylation of HNF4alpha leads to a reduced trans-activational capacity of the nuclear receptor in luciferase reporter gene assay. We confirm the functional relevance of these findings by demonstrating with ChIP-qPCR experiments that 30-minute activation of ERK1/2 leads to reduced chromatin binding of HNF4alpha. Accordingly, we have observed decreasing but not disappearing binding of HNF4alpha to the target genes. In addition, 24-hour activation of the pathway further decreased HNF4alpha chromatin binding to specific loci in ChIP-qPCR experiments, which confirms the previous reports on the decreased expression of the HNF4a gene due to ERK1/2 activation. Our data suggest that the ERK1/2 pathway plays an important role in the regulation of HNF4alpha-dependent hepatic gene expression

Cortactin Tyrosine Phosphorylation Promotes Its Deacetylation and Inhibits Cell Spreading

Background: Cortactin is a classical Src kinase substrate that participates in actin cytoskeletal dynamics by activating the Arp2/3 complex and interacting with other regulatory proteins, including FAK. Cortactin has various domains that may contribute to the assembly of different protein platforms to achieve process specificity. Though the protein is known to be regulated by post-translational modifications such as phosphorylation and acetylation, how tyrosine phosphorylation regulates cortactin activity is poorly understood. Since the basal level of tyrosine phosphorylation is low, this question must be studied using stimulated cell cultures, which are physiologically relevant but unreliable and difficult to work with. In fact, their unreliability may be the cause of some contradictory findings about the dynamics of tyrosine phosphorylation of cortactin in different processes. Methodology/Principal Findings: In the present study, we try to overcome these problems by using a Functional Interaction Trap (FIT) system, which involves cotransfecting cells with a kinase (Src) and a target protein (cortactin), both of which are fused to complementary leucine-zipper domains. The FIT system allowed us to control precisely the tyrosine phosphorylation of cortactin and explore its relationship with cortactin acetylation. Conclusions/Significance: Using this system, we provide definitive evidence that a competition exists between acetylation and tyrosine phosphorylation of cortactin and that phosphorylation inhibits cell spreading. We confirmed the results fro

Gefitinib Induces Epidermal Growth Factor Receptor Dimers Which Alters the Interaction Characteristics with 125I-EGF

The tyrosine kinase inhibitor gefitinib inhibits growth in some tumor types by targeting the epidermal growth factor receptor (EGFR). Previous studies show that the affinity of the EGF-EGFR interaction varies between hosting cell line, and that gefitinib increases the affinity for some cell lines. In this paper, we investigate possible mechanisms behind these observations. Real-time interaction analysis in LigandTracer® Grey revealed that the HER2 dimerization preventing antibody pertuzumab clearly modified the binding of 125I-EGF to EGFR on HER2 overexpressing SKOV3 cells in the presence of gefitinib. Pertuzumab did not affect the binding on A431 cells, which express low levels of HER2. Cross-linking measurements showed that gefitinib increased the amount of EGFR dimers 3.0–3.8 times in A431 cells in the absence of EGF. In EGF stimulated SKOV3 cells the amount of EGFR dimers increased 1.8–2.2 times by gefitinib, but this effect was cancelled by pertuzumab. Gefitinib treatment did not alter the number of EGFR or HER2 expressed in tumor cell lines A431, U343, SKOV3 and SKBR3. Real-time binding traces were further analyzed in a novel tool, Interaction Map, which deciphered the different components of the measured interaction and supports EGF binding to multiple binding sites. EGFR and HER2 expression affect the levels of EGFR monomers, homodimers and heterodimers and EGF binds to the various monomeric/dimeric forms of EGFR with unique binding properties. Taken together, we conclude that dimerization explains the varying affinity of EGF – EGFR in different cells, and we propose that gefitinib induces EGFR dimmers, which alters the interaction characteristics with 125I-EGF

The Adaptor Molecule Nck Localizes the WAVE Complex to Promote Actin Polymerization during CEACAM3-Mediated Phagocytosis of Bacteria

Background: CEACAM3 is a granulocyte receptor mediating the opsonin-independent recognition and phagocytosis of human-restricted CEACAM-binding bacteria. CEACAM3 function depends on an intracellular immunoreceptor tyrosine-based activation motif (ITAM)-like sequence that is tyrosine phosphorylated by Src family kinases upon receptor engagement. The phosphorylated ITAM-like sequence triggers GTP-loading of Rac by directly associating with the guanine nucleotide exchange factor (GEF) Vav. Rac stimulation in turn is critical for actin cytoskeleton rearrangements that generate lamellipodial protrusions and lead to bacterial uptake. Principal Findings: In our present study we provide biochemical and microscopic evidence that the adaptor proteins Nck1 and Nck2, but not CrkL, Grb2 or SLP-76, bind to tyrosine phosphorylated CEACAM3. The association is phosphorylation-dependent and requires the Nck SH2 domain. Overexpression of the isolated Nck1 SH2 domain, RNAi-mediated knock-down of Nck1, or genetic deletion of Nck1 and Nck2 interfere with CEACAM3-mediated bacterial internalization and with the formation of lamellipodial protrusions. Nck is constitutively associated with WAVE2 and directs the actin nucleation promoting WAVE complex to tyrosine phosphorylated CEACAM3. In turn, dominant-negative WAVE2 as well as shRNA-mediated knock-down of WAVE2 or the WAVE-complex component Nap1 reduce internalization of bacteria. Conclusions: Our results provide novel mechanistic insight into CEACAM3-initiated phagocytosis. We suggest that the CEACAM3 ITAM-like sequence is optimized to co-ordinate a minimal set of cellular factors needed to efficiently trigger actin-based lamellipodial protrusions and rapid pathogen engulfment

New Insight into the Transcarbamylase Family: The Structure of Putrescine Transcarbamylase, a Key Catalyst for Fermentative Utilization of Agmatine

Transcarbamylases reversibly transfer a carbamyl group from carbamylphosphate (CP) to an amine. Although aspartate transcarbamylase and ornithine transcarbamylase (OTC) are well characterized, little was known about putrescine transcarbamylase (PTC), the enzyme that generates CP for ATP production in the fermentative catabolism of agmatine. We demonstrate that PTC (from Enterococcus faecalis), in addition to using putrescine, can utilize L-ornithine as a poor substrate. Crystal structures at 2.5 Å and 2.0 Å resolutions of PTC bound to its respective bisubstrate analog inhibitors for putrescine and ornithine use, N-(phosphonoacetyl)-putrescine and δ-N-(phosphonoacetyl)-L-ornithine, shed light on PTC preference for putrescine. Except for a highly prominent C-terminal helix that projects away and embraces an adjacent subunit, PTC closely resembles OTCs, suggesting recent divergence of the two enzymes. Since differences between the respective 230 and SMG loops of PTC and OTC appeared to account for the differential preference of these enzymes for putrescine and ornithine, we engineered the 230-loop of PTC to make it to resemble the SMG loop of OTCs, increasing the activity with ornithine and greatly decreasing the activity with putrescine. We also examined the role of the C-terminal helix that appears a constant and exclusive PTC trait. The enzyme lacking this helix remained active but the PTC trimer stability appeared decreased, since some of the enzyme eluted as monomers from a gel filtration column. In addition, truncated PTC tended to aggregate to hexamers, as shown both chromatographically and by X-ray crystallography. Therefore, the extra C-terminal helix plays a dual role: it stabilizes the PTC trimer and, by shielding helix 1 of an adjacent subunit, it prevents the supratrimeric oligomerizations of obscure significance observed with some OTCs. Guided by the structural data we identify signature traits that permit easy and unambiguous annotation of PTC sequences