scholarly journals Requirements of Multiple Domains of SLI-1, aCaenorhabditis elegansHomologue of c-Cbl, and an Inhibitory Tyrosine in LET-23 in Regulating Vulval Differentiation

2000 ◽  
Vol 11 (11) ◽  
pp. 4019-4031 ◽  
Author(s):  
Charles H. Yoon ◽  
Chieh Chang ◽  
Neil A. Hopper ◽  
Giovanni M. Lesa ◽  
Paul W. Sternberg
Keyword(s):  
Calcium Binding ◽  
Ring Finger ◽  
Growth Factor Receptor ◽  
Chimeric Protein ◽  
Sh2 Domain ◽  
Negative Regulator ◽  
Functional Assay ◽  
Ring Finger Domain ◽  
C Terminus ◽  
Ras Activation

SLI-1, a Caenorhabditis elegans homologue of the proto-oncogene product c-Cbl, is a negative regulator of LET-23-mediated vulval differentiation. Lack of SLI-1 activity can compensate for decreased function of the LET-23 epidermal growth factor receptor, the SEM-5 adaptor, but not the LET-60 RAS, suggesting that SLI-1 acts before RAS activation. SLI-1 and c-Cbl comprise an N-terminal region (termed SLI-1:N/Cbl-N, containing a four-helix bundle, an EF hand calcium-binding domain, and a divergent SH2 domain) followed by a RING finger domain and a proline-rich C-terminus. In a transgenic functional assay, the proline-rich C-terminal domain is not essential for sli-1(+) function. A protein lacking the SH2 and RING finger domains has no activity, but a chimeric protein with the SH2 and RING finger domains of SLI-1 replaced by the equivalent domains of c-Cbl has activity. The RING finger domain of c-Cbl has been shown recently to enhance ubiquitination of active RTKs by acting as an E3 ubiquitin–protein ligase. We find that the RING finger domain of SLI-1 is partially dispensable. Further, we identify an inhibitory tyrosine of LET-23 requiring sli-1(+) for its effects: removal of this tyrosine closely mimics the loss ofsli-1 but not of another negative regulator,ark-1. Thus, we suggest that this inhibitory tyrosine mediates its effects through SLI-1, which in turn inhibits signaling upstream of LET-60 RAS in a manner not wholly dependent on the ubiquitin–ligase domain.

Binding of the Src SH2 domain to phosphopeptides is determined by residues in both the SH2 domain and the phosphopeptides

1993 ◽  
Vol 13 (12) ◽  
pp. 7278-7287
Author(s):  
K B Bibbins ◽  
H Boeuf ◽  
H E Varmus
Keyword(s):  
Intramolecular Interaction ◽  
Growth Factor Receptor ◽  
Sh2 Domain ◽  
Binding Properties ◽  
Vitro Assay ◽  
Sh2 Domains ◽  
Free Peptide ◽  
C Terminus

Src homology 2 (SH2) domains are found in a variety of signaling proteins and bind phosphotyrosine-containing peptide sequences. To explore the binding properties of the SH2 domain of the Src protein kinase, we used immobilized phosphopeptides to bind purified glutathione S-transferase-Src SH2 fusion proteins. With this assay, as well as a free-peptide competition assay, we have estimated the affinities of the Src SH2 domain for various phosphopeptides relative to a Src SH2-phosphopeptide interaction whose Kd has been determined previously (YEEI-P; Kd = 4 nM). Two Src-derived phosphopeptides, one containing the regulatory C-terminal Tyr-527 and another containing the autophosphorylation site Tyr-416, bind the Src SH2 domain in a specific though low-affinity manner (with about 10(4)-lower affinity than the YEEI-P peptide). A platelet-derived growth factor receptor (PDGF-R) phosphopeptide containing Tyr-857 does not bind appreciably to the Src SH2 domain, suggesting it is not the PDGF-R binding site for Src as previously reported. However, another PDGF-R-derived phosphopeptide containing Tyr-751 does bind the Src SH2 domain (with an affinity approximately 2 orders of magnitude lower than that of YEEI-P). All of the phosphopeptides which bind to the Src SH2 domain contain a glutamic acid at position -3 or -4 with respect to phosphotyrosine; changing this residue to alanine greatly diminishes binding. We have also tested Src SH2 mutants for their binding properties and have interpreted our results in light of the recent crystal structure solution for the Src SH2 domain. Mutations in various conserved and nonconserved residues (R155A, R155K, N198E, H201R, and H201L) cause slight reductions in binding, while two mutations cause severe reductions. The W148E mutant domain, which alters the invariant tryptophan that marks the N-terminal border of the SH2 domain, binds poorly to phosphopeptides. Inclusion of the SH3 domain in the fusion protein partially restores the binding by the W148E mutant. A change in the invariant arginine that coordinates twice with phosphotyrosine in the peptide (R175L) results in a nearly complete loss of binding. The R175L mutant does display high affinity for the PDGF-R peptide containing Tyr-751, via an interaction that is at least partly phosphotyrosine independent. We have used this interaction to show that the R175L mutation also disrupts the intramolecular interaction between the Src SH2 domain and the phosphorylated C terminus within the context of the entire Src protein; thus, the binding properties observed for mutant domains in an in vitro assay appear to mimic those that occur in vivo.

scholarly journals Zinc Binding Properties of Engineered RING Finger Domain of Arkadia E3 Ubiquitin Ligase

2010 ◽  
Vol 2010 ◽  
pp. 1-7 ◽  
Author(s):  
Christos T. Chasapis ◽  
Ariadni K. Loutsidou ◽  
Malvina G. Orkoula ◽  
Georgios A. Spyroulias
Keyword(s):  
Ubiquitin Ligase ◽  
Structural Integrity ◽  
Recombinant Expression ◽  
E3 Ubiquitin Ligase ◽  
Ring Finger ◽  
Ring Domain ◽  
Site Directed Mutagenesis ◽  
Zinc Binding ◽  
Ring Finger Domain ◽  
C Terminus

Human Arkadia is a nuclear protein consisted of 989 amino acid residues, with a characteristic RING domain in its C-terminus. The RING domain harbours the E3 ubiquitin ligase activity needed by Arkadia to ubiquitinate its substrates such as negative regulators of TGF- signaling. The RING finger domain of Arkadia is a RING-H2 type and its structure and stability is strongly dependent on the presence of two bound Zn(II) ions attached to the protein frame through a defined Cys3-His2-Cys3 motif. In the present paper we transform the RING-H2 type of Arkadia finger domain to nonnative RING sequence, substituting the zinc-binding residues or to Arginine, through site-directed mutagenesis. The recombinant expression, inEscherichia coli, of the mutants C955R and H960R reveal significant lower yield in respect with the native polypeptide of Arkadia RING-H2 finger domain. In particular, only the C955R mutant exhibits expression yield sufficient for recombinant protein isolation and preliminary studies. Atomic absorption measurements and preliminary NMR data analysis reveal that the C955R point mutation in the RING Finger domain of Arkadia diminishes dramatically the zinc binding affinity, leading to the breakdown of the global structural integrity of the RING construct.

scholarly journals EGFRAP encodes a new negative regulator of the EGFR acting in both normal and oncogenic EGFR/Ras-driven tissue morphogenesis

PLoS Genetics ◽  
2021 ◽  
Vol 17 (8) ◽  
pp. e1009738
Author(s):  
Jennifer Soler Beatty ◽  
Cristina Molnar ◽  
Carlos M. Luque ◽  
Jose F. de Celis ◽  
María D. Martín-Bermudo
Keyword(s):  
Imaginal Disc ◽  
Feedback Loop ◽  
Notch Pathway ◽  
Sh2 Domain ◽  
Negative Regulator ◽  
Ras Signaling ◽  
Ras Pathway ◽  
Phosphorylated Form ◽  
Ras Activation ◽  
Surveillance Mechanism

Activation of Ras signaling occurs in ~30% of human cancers. However, activated Ras alone is insufficient to produce malignancy. Thus, it is imperative to identify those genes cooperating with activated Ras in driving tumoral growth. In this work, we have identified a novel EGFR inhibitor, which we have named EGFRAP, for EGFR adaptor protein. Elimination of EGFRAP potentiates activated Ras-induced overgrowth in the Drosophila wing imaginal disc. We show that EGFRAP interacts physically with the phosphorylated form of EGFR via its SH2 domain. EGFRAP is expressed at high levels in regions of maximal EGFR/Ras pathway activity, such as at the presumptive wing margin. In addition, EGFRAP expression is up-regulated in conditions of oncogenic EGFR/Ras activation. Normal and oncogenic EGFR/Ras-mediated upregulation of EGRAP levels depend on the Notch pathway. We also find that elimination of EGFRAP does not affect overall organogenesis or viability. However, simultaneous downregulation of EGFRAP and its ortholog PVRAP results in defects associated with increased EGFR function. Based on these results, we propose that EGFRAP is a new negative regulator of the EGFR/Ras pathway, which, while being required redundantly for normal morphogenesis, behaves as an important modulator of EGFR/Ras-driven tissue hyperplasia. We suggest that the ability of EGFRAP to functionally inhibit the EGFR pathway in oncogenic cells results from the activation of a feedback loop leading to increase EGFRAP expression. This could act as a surveillance mechanism to prevent excessive EGFR activity and uncontrolled cell growth.

scholarly journals Slap Negatively Regulates Src Mitogenic Function but Does Not Revert Src-Induced Cell Morphology Changes

2000 ◽  
Vol 20 (10) ◽  
pp. 3396-3406 ◽  
Author(s):  
Gaël Manes ◽  
Paul Bello ◽  
Serge Roche
Keyword(s):  
Dna Synthesis ◽  
Cell Transformation ◽  
Sh2 Domain ◽  
Fiber Formation ◽  
Dominant Negative ◽  
Negative Regulator ◽  
C Terminus ◽  
Factor C

ABSTRACT Src-like adapter protein (Slap) is a recently identified protein that negatively regulates mitogenesis in murine fibroblasts (S. Roche, G. Alonso, A. Kazlausakas, V. M. Dixit, S. A. Courtneidge, and A. Pandey, Curr. Biol. 8:975–978, 1998) and comprises an SH3 and SH2 domain with striking identity to the corresponding Src domains. In light of this, we sought to investigate whether Slap could be an antagonist of all Src functions. Like Src, Slap was found to be myristylated in vivo and largely colocalized with Src when coexpressed in Cos7 cells. Microinjection of a Slap-expressing construct into quiescent NIH 3T3 cells inhibited platelet-derived growth factor (PDGF)-induced DNA synthesis, and the inhibition was rescued by the transcription factor c-Myc but not by c-Jun/c-Fos expression. Fyn (or Src) overexpression overrides the G1/S block induced by both SrcK− and a Slap mutant with a deletion of its C terminus (SlapΔC), but not the block induced by Slap or SlapΔSH3, implying that the C terminus is a noncompetitive inhibitor of Src mitogenic function. Furthermore, a chimeric adapter comprising SrcΔK fused to the Slap C terminus (Src/SlapC) also inhibited Src function during the PDGF response in a noncompetitive manner, as Src coexpression could not rescue PDGF signaling. Slap, however, did not reverse deregulated Src-induced cell transformation, as it was unable to inhibit depolymerization of actin stress fibers while still being able to inhibit SrcY527F-induced DNA synthesis. This was attributed to a distinct Slap SH3 binding specificity, since the chimeric Slap/SrcSH3 molecule, in which the Slap SH3 was replaced by the Src SH3 sequence, substantially restored stress fiber formation. Indeed, three amino acids important for ligand binding in Src SH3 were replaced in the Slap SH3 sequence; Slap SH3 did not bind to the Src SH3 partners p85α, Shc, and Sam68 in vitro, and the chimeric tyrosine kinase Slap/SrcK, composed of SlapΔC fused to the SH2 linker kinase sequence of Src, was not regulated in vivo. Furthermore, the Src SH3 domain is required for signaling during mitogenesis and since Slap/SrcK behaved as a dominant negative in the PDGF mitogenic response when microinjected into quiescent fibroblasts. We conclude that Slap is a negative regulator of Src during mitogenesis involving both the SH2 and the C terminus domains in a noncompetitive manner, but it does not regulate all Src function due to specific SH3 binding substrates.

scholarly journals Critical Contribution of the MDM2 Acidic Domain to p53 Ubiquitination

2003 ◽  
Vol 23 (14) ◽  
pp. 4939-4947 ◽  
Author(s):  
Hidehiko Kawai ◽  
Dmitri Wiederschain ◽  
Zhi-Min Yuan
Keyword(s):  
Nuclear Export ◽  
Critical Role ◽  
Ring Finger ◽  
E3 Ligase ◽  
Chimeric Protein ◽  
Chimeric Proteins ◽  
Ring Finger Domain ◽  
Acidic Domain ◽  
Ubiquitin E3 Ligase

ABSTRACT MDM2 is an E3 ubiquitin ligase that targets p53 for proteasomal degradation. Recent studies have shown, however, that the ring-finger domain (RFD) of MDM2, where the ubiquitin E3 ligase activity resides, is necessary but not sufficient for p53 ubiquitination, suggesting that an additional activity of MDM2 might be required. To test this possibility, we generated a series of MDM2/MDMX chimeric proteins to assess the contribution of each domain of MDM2 to the ubiquitination process. MDMX is a close structural homolog of MDM2 that nevertheless lacks the E3 ligase activity in vivo. We demonstrate here that MDMX gains self-ubiquitination activity and becomes extremely unstable upon introduction of the MDM2 RFD, indicating that the RFD is essential for self-ubiquitination. This MDMX chimeric protein, however, is unable to ubiquitinate p53 in vivo despite its E3 ligase activity and binding to p53, separating the self-ubiquitination activity of MDM2 from its ability to ubiquitinate p53. Significantly, fusion of the central acidic domain (AD) of MDM2 to the MDMX chimeric protein renders the protein fully capable of ubiquitinating p53, and p53 ubiquitination is associated with p53 degradation and nuclear export. Moreover, the AD mini protein expressed in trans can functionally rescue the AD-lacking MDM2 mutant, further supporting a critical role for the AD in MDM2-mediated p53 ubiquitination.

A novel negative regulatory function of the phosphoprotein associated with glycosphingolipid-enriched microdomains: blocking Ras activation

Blood ◽  
2007 ◽  
Vol 110 (2) ◽  
pp. 596-625 ◽  
Author(s):  
Michal Smida ◽  
Anita Posevitz-Fejfar ◽  
Vaclav Horejsi ◽  
Burkhart Schraven ◽  
Jonathan A. Lindquist
Keyword(s):  
T Cells ◽  
T Cell Activation ◽  
Cell Activation ◽  
Kinase Activity ◽  
Src Kinase ◽  
Sh2 Domain ◽  
Negative Regulator ◽  
Regulatory Function ◽  
Ras Activation ◽  
Anergic T Cells

Abstract In primary human T cells, anergy induction results in enhanced p59Fyn activity. Because Fyn is the kinase primarily responsible for the phosphorylation of PAG (the phosphoprotein associated with glycosphingolipid-enriched microdomains), which negatively regulates Src-kinase activity by recruiting Csk (the C-terminal Src kinase) to the membrane, we investigated whether anergy induction also affects PAG. Analysis of anergic T cells revealed that PAG is hyperphosphorylated at the Csk binding site, leading to enhanced Csk recruitment and inhibitory tyrosine phosphorylation within Fyn. This together with enhanced phosphorylation of a tyrosine within the SH2 domain of Fyn leads to the formation of a hyperactive conformation, thus explaining the enhanced Fyn kinase activity. In addition, we have also identified the formation of a multiprotein complex containing PAG, Fyn, Sam68, and RasGAP in stimulated T cells. We demonstrate that PAG-Fyn overexpression is sufficient to suppress Ras activation in Jurkat T cells and show that this activity is independent of Csk binding. Thus, in addition to negatively regulating Src family kinases by recruiting Csk, PAG also negatively regulates Ras by recruiting RasGAP to the membrane. Finally, by knocking down PAG, we demonstrate both enhanced Src kinase activity and Ras activation, thereby establishing PAG as an important negative regulator of T-cell activation.

scholarly journals Binding of the Src SH2 domain to phosphopeptides is determined by residues in both the SH2 domain and the phosphopeptides.

1993 ◽  
Vol 13 (12) ◽  
pp. 7278-7287 ◽  
Author(s):  
K B Bibbins ◽  
H Boeuf ◽  
H E Varmus
Keyword(s):  
Intramolecular Interaction ◽  
Growth Factor Receptor ◽  
Sh2 Domain ◽  
Binding Properties ◽  
Vitro Assay ◽  
Sh2 Domains ◽  
Free Peptide ◽  
C Terminus

Src homology 2 (SH2) domains are found in a variety of signaling proteins and bind phosphotyrosine-containing peptide sequences. To explore the binding properties of the SH2 domain of the Src protein kinase, we used immobilized phosphopeptides to bind purified glutathione S-transferase-Src SH2 fusion proteins. With this assay, as well as a free-peptide competition assay, we have estimated the affinities of the Src SH2 domain for various phosphopeptides relative to a Src SH2-phosphopeptide interaction whose Kd has been determined previously (YEEI-P; Kd = 4 nM). Two Src-derived phosphopeptides, one containing the regulatory C-terminal Tyr-527 and another containing the autophosphorylation site Tyr-416, bind the Src SH2 domain in a specific though low-affinity manner (with about 10(4)-lower affinity than the YEEI-P peptide). A platelet-derived growth factor receptor (PDGF-R) phosphopeptide containing Tyr-857 does not bind appreciably to the Src SH2 domain, suggesting it is not the PDGF-R binding site for Src as previously reported. However, another PDGF-R-derived phosphopeptide containing Tyr-751 does bind the Src SH2 domain (with an affinity approximately 2 orders of magnitude lower than that of YEEI-P). All of the phosphopeptides which bind to the Src SH2 domain contain a glutamic acid at position -3 or -4 with respect to phosphotyrosine; changing this residue to alanine greatly diminishes binding. We have also tested Src SH2 mutants for their binding properties and have interpreted our results in light of the recent crystal structure solution for the Src SH2 domain. Mutations in various conserved and nonconserved residues (R155A, R155K, N198E, H201R, and H201L) cause slight reductions in binding, while two mutations cause severe reductions. The W148E mutant domain, which alters the invariant tryptophan that marks the N-terminal border of the SH2 domain, binds poorly to phosphopeptides. Inclusion of the SH3 domain in the fusion protein partially restores the binding by the W148E mutant. A change in the invariant arginine that coordinates twice with phosphotyrosine in the peptide (R175L) results in a nearly complete loss of binding. The R175L mutant does display high affinity for the PDGF-R peptide containing Tyr-751, via an interaction that is at least partly phosphotyrosine independent. We have used this interaction to show that the R175L mutation also disrupts the intramolecular interaction between the Src SH2 domain and the phosphorylated C terminus within the context of the entire Src protein; thus, the binding properties observed for mutant domains in an in vitro assay appear to mimic those that occur in vivo.

scholarly journals c-Cbl targets PD-1 in immune cells for proteasomal degradation and modulates colorectal tumor growth

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Chimera Lyle ◽  
Sean Richards ◽  
Kei Yasuda ◽  
Marc Arthur Napoleon ◽  
Joshua Walker ◽  
...  
Keyword(s):  
Tumor Microenvironment ◽  
Immune Cells ◽  
Immune Checkpoint ◽  
Ring Finger ◽  
Neutralizing Antibody ◽  
Proteasomal Degradation ◽  
Negative Regulator ◽  
C Terminus ◽  
Fold Reduction ◽  
Programmed Cell Death 1

AbstractCasitas B lymphoma (c-Cbl) is an E3 ubiquitin ligase and a negative regulator of colorectal cancer (CRC). Despite its high expression in immune cells, the effect of c-Cbl on the tumor microenvironment remains poorly understood. Here we demonstrate that c-Cbl alters the tumor microenvironment and suppresses Programmed cell death-1 (PD-1) protein, an immune checkpoint receptor. Using syngeneic CRC xenografts, we observed significantly higher growth of xenografts and infiltrating immune cells in c-Cbl+/− compared to c-Cbl+/+ mice. Tumor-associated CD8+ T-lymphocytes and macrophages of c-Cbl+/− mice showed 2–3-fold higher levels of PD-1. Functionally, macrophages from c-Cbl+/− mice showed a 4–5-fold reduction in tumor phagocytosis, which was restored with an anti-PD-1 neutralizing antibody suggesting regulation of PD-1 by c-Cbl. Further mechanistic probing revealed that C-terminus of c-Cbl interacted with the cytoplasmic tail of PD-1. c-Cbl destabilized PD-1 through ubiquitination- proteasomal degradation depending on c-Cbl’s RING finger function. This data demonstrates c-Cbl as an E3 ligase of PD-1 and a regulator of tumor microenvironment, both of which were unrecognized components of its tumor suppressive activity. Advancing immune checkpoint and c-Cbl biology, our study prompts for probing of PD-1 regulation by c-Cbl in conditions driven by immune checkpoint abnormalities such as cancers and autoimmune diseases.

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