>>Does this paper and its placement support the claimed leadership? <<
Still a good question. The website shows Rigel's various ubiquitin ligase associated candidates in the screening process. So preclinical stuff is yet to come, at least in print. However, no one else seems to be much farther along. Humorously, a couple of teams claimed the identification of a "novel" Src-like adaptor protein, SLAP-2 (which associates with the E3 ubiquitin ligase Cbl), a good six months after Rigel found it in 2001 . . .
>>J Biol Chem. 2002 May 24;277(21):19131-8. Epub 2002 Mar 12.
A novel Src homology 2 domain-containing molecule, Src-like adapter protein-2 (SLAP-2), which negatively regulates T cell receptor signaling.
Pandey A, Ibarrola N, Kratchmarova I, Fernandez MM, Constantinescu SN, Ohara O, Sawasdikosol S, Lodish HF, Mann M.
Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142, USA.
We have cloned a novel adapter protein containing Src homology 2 and Src homology 3 domains similar to the Src family of tyrosine kinases. This molecule lacks a catalytic tyrosine kinase domain and is related to a previously identified protein, Src-like adapter protein (SLAP), and is therefore designated SLAP-2. Northern blot analysis indicates that SLAP-2 is predominantly expressed in the immune system. Jurkat T cells express SLAP-2 protein and overexpression of SLAP-2 in these cells negatively regulates T cell receptor signaling as assessed by interleukin-2 promoter or NF-AT promoter reporter constructs. Mutational analysis revealed that an intact SH2 domain of SLAP-2 is essential for this inhibitory effect, whereas mutation of the SH3 domain alone has no effect. This inhibitory effect is upstream of the activation of Ras and increase of intracellular calcium levels, as no inhibition was observed when the cells were activated by phorbol ester plus ionomycin. SLAP-2 interacts with Cbl in vivo in a phosphorylation independent manner and with ZAP-70 and T cell receptor zeta chain upon T cell receptor activation. Finally, we show that the mutation of a predicted myristoylation site within the NH(2)-terminal of SLAP-2 is essential for its inhibitory effect. This report therefore implicates SLAP and SLAP-2 as a family of adapter proteins that negatively regulate T cell receptor signaling.<<
>>Mol Cell Biol. 2002 Jun;22(12):4241-55.
Functional cooperation between c-Cbl and Src-like adaptor protein 2 in the negative regulation of T-cell receptor signaling.
Loreto MP, Berry DM, McGlade CJ.
Department of Medical Biophysics, University of Toronto and Arthur and Sonia Labatt Brain Tumour Research Centre, The Hospital for Sick Children, Toronto, Ontario, Canada M5G 1X8.
Adaptor proteins assemble multiprotein signaling complexes, enabling the transduction of intracellular signals. While many adaptor proteins positively regulate signaling in this manner, a subgroup of adaptors function as negative regulators. Here we report the identification of a hematopoiesis-specific adaptor protein that we have designated Src-like adaptor protein 2 (SLAP-2). SLAP-2 is most closely related to SLAP and contains a Src homology 3 (SH3) domain and an SH2 domain, as well as an amino-terminal myristoylation site that mediates SLAP-2 association with membranes. Following stimulation of primary thymocytes with anti-CD3 and anti-CD28, SLAP-2 coimmunoprecipitates with tyrosine-phosphorylated c-Cbl and an unidentified protein of approximately 72 kDa. In activated Jurkat T cells, SLAP-2 also binds an additional 70-kDa phosphoprotein, identified as ZAP-70. Binding of SLAP-2 to both p72 and ZAP-70 is dependent on its SH2 domain, while c-Cbl interacts with the carboxy-terminal region. Overexpression of wild-type SLAP-2 alone or in combination with c-Cbl in Jurkat T cells leads to inhibition of T-cell antigen receptor-induced activation of nuclear factor of activated T cells. The inhibitory effect of SLAP-2 requires the carboxy-terminal c-Cbl binding region. Expression of SLAP-2 with SYK or ZAP-70 in COS cells or Jurkat T cells causes the degradation of these kinases, and SLAP-2 overexpression in Jurkat T cells reduces the surface expression of CD3. These results suggest that the mechanism of action of SLAP-2 and the related protein SLAP is to promote c-Cbl-dependent degradation of the tyrosine kinases SYK and ZAP-70 and down-regulation of CD3 at the cell surface.<<
Message 16707938
In the following post, Rick said if we found any mouse data to please yell. I don't know if this really qualifies, as it does not directly involve a Rigel molecule -- though it might be closely related, so . . .
>>Carcinogenesis. 2004 Feb;25(2):157-167. Epub 2003 Oct 24.
RING protein Trim32 associated with skin carcinogenesis has anti-apoptotic and E3-ubiquitin ligase properties.
Horn EJ, Albor A, Liu Y, El-Hizawi S, Vanderbeek GE, Babcock M, Bowden GT, Hennings H, Lozano G, Weinberg WC, Kulesz-Martin M.
Department of Dermatology and Department of Cell and Developmental Biology, Oregon Health and Science University, Portland, OR 97239, USA, 2Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, State University of New York at Buffalo, Buffalo, NY 14263, USA, 3Department of Radiation Oncology, University of Arizona, Tucson, AZ 85724, USA, 4Laboratory of Cellular Carcinogenesis and Tumor Promotion, National Cancer Institute/National Institutes of Health, Bethesda, MD 20892, USA, 5Department of Molecular Genetics, The University of Texas M.D. Anderson Cancer Center Houston, TX 77030, USA and 6Laboratory of Immunobiology, US Food and Drug Administration, Rockville, MD 20857, USA.
Tripartite motif protein 32, Trim32, mRNA and protein expression was elevated in independently transformed and tumorigenic keratinocytes of a mouse epidermal carcinogenesis model, in ultraviolet B (UVB)-induced squamous cell carcinomas (SCC), and in approximately 20-25% of chemically induced mouse papillomas and human head and neck SCCs. This suggests that elevated Trim32 expression occurs frequently in experimental epidermal carcinogenesis and is relevant to human cancer. Transduced Trim32 increased colony number in an epidermal in vitro transformation assay and epidermal thickening in vivo when skin-grafted to athymic nu/nu mice. These effects were not associated with proliferation and were not sufficient for tumorigenesis, even with 12-O-tetradecanoylphorbol-13-acetate treatment or defects in the tumor suppressor p53. However, transduced Trim32 inhibited the synergistic effect of tumor necrosis factor alpha (TNFalpha) on UVB-induced apoptosis of keratinocytes in vitro and the apoptotic response of keratinocyte grafts exposed to UVB-light in vivo. Consistent with its RING domain, Trim32 exhibited characteristics of E3-ubiquitin ligases, including being ubiquitylated itself and interacting with ubiquitylated proteins, with increases in these properties following treatment of cultured keratinocytes with TNFalpha/UVB. Interestingly, missense point mutation of human TRIM32 has been reported in Limb-Girdle Muscular Dystrophy Type 2H, an autosomal recessive disease. We propose a model in which Trim32 activities as an E3-ubiquitin ligase favor initiated cell survival in carcinogenesis by blocking UVB-induced TNFalpha apoptotic signaling.<<
>>Ubiquitin ligases...... I don't know much about them. If there are different ligases for regulatory proteins like CDKs (take note, apostrophe police!!), then it might make sense. If not, it seems like just another anti-cancer approach that will land us in "selective toxicity" land. If anyone runs across any preclinical (mouse, etc.) data, please yell! <<
It would appear that there are?
>>Proc Natl Acad Sci U S A. 2003 Sep 2;100(18):10231-6. Epub 2003 Aug 18.
Degradation of p57Kip2 mediated by SCFSkp2-dependent ubiquitylation.
Kamura T, Hara T, Kotoshiba S, Yada M, Ishida N, Imaki H, Hatakeyama S, Nakayama K, Nakayama KI.
Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, Fukuoka 812-8582, Japan.
The abundance of the cyclin-dependent kinase (CDK) inhibitor p57Kip2, an important regulator of cell cycle progression, is thought to be controlled by the ubiquitin-proteasome pathway. The Skp1/Cul1/F-box (SCF)-type E3 ubiquitin ligase complex SCFSkp2 has now been shown to be responsible for regulating the cellular level of p57Kip2 by targeting it for ubiquitylation and proteolysis. The elimination of p57Kip2 was impaired in Skp2-/- cells, resulting in abnormal accumulation of the protein. Coimmunoprecipitation analysis also revealed that Skp2 interacts with p57Kip2 in vivo. Overexpression of WT Skp2 promoted degradation of p57Kip2, whereas expression of a dominant negative mutant of Skp2 prolonged the half-life of p57Kip2. Mutation of the threonine residue (Thr-310) of human p57Kip2 that is conserved between the COOH-terminal QT domains of p57Kip2 and p27Kip1 prevented the effect of Skp2 on the stability of p57Kip2, suggesting that phosphorylation at this site is required for SCFSkp2-mediated ubiquitylation. Finally, the purified recombinant SCFSkp2 complex mediated p57Kip2 ubiquitylation in vitro in a manner dependent on the presence of the cyclin E-CDK2 complex. These observations thus demonstrate that the SCFSkp2 complex plays an important role in cell-cycle progression by determining the abundance of p57Kip2 and that of the related CDK inhibitor p27Kip1.<<
Cheers, Tuck |
