TcR ͞CD3 ligation initiates a signaling cascade involving CD4͞CD8-p56 lck , p59 fyn , and ZAP-70, as well as lymphoid downstream proteins VAV, SLP-76, and FYB͞SLAP. A current question concerns the nature of the downstream binding partner(s) of FYB in T cells. In this study, using a two-hybrid screen with FYB as bait, we have identified eight clones, four of which correspond to the recently published lymphoid protein SKAP55, and two which correspond to a related protein with some 44% homology to SKAP55 (termed SKAP55-related protein, SKAP55R). The SKAP55 clones showed only minor differences (two substitutions and one residue deletion) from SKAP55. SKAP55R has the same overall structure as SKAP55 except for the presence of a unique N terminus with a well-defined coiled-coil domain. Both SKAP55 and SKAP55R were found to bind FYB through their SH3 domains and to act as substrates for the FYN kinase in T cells. Furthermore, immunof luorescence confocal microscopy showed that FYB and SKAP55 colocalize in the perinuclear region of cells. SKAP55 also colocalizes with another FYB binding protein, SLP-76. Taken together, these observations demonstrate that FYB is part of an interactive matrix with SKAP55 and a SKAP55-related protein.Ligation of CD4͞CD8-p56 lck and the T cell receptor complex (TcR ͞CD3) activates src protein-tyrosine kinases (PTKs) p56 lck and p59 fyn (1), leading to phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) of the TcR and CD3 chains (2-4). The phosphorylated ITAM recruits ZAP-70 by means of tandem SH2 domain binding (5, 6). CD4-p56 lck can further up-regulate ZAP-70 catalytic activity by phosphorylation of residue Y-493 (7). The importance of ZAP-70 in T cell signaling has been shown by defects in early signaling events and interleukin 2 (IL-2) transcription in ZAP-70-negative Jurkat cells (8). The Lck-SH2 domain can also bind to ZAP-70, thereby consolidating the CD4͞CD8-p56 lck and TcR ͞CD3 aggregate (9). Consistent with this capability, p56 lck has been found associated with the TcR ͞ CD3 complex (10). p56 lck may also play roles in signaling by other systems by virtue of its ability to phosphorylate other surface receptors such as CD5 and CD28 (11-13).Recent advances have identified downstream targets of the TcR͞CD3 and CD4͞CD8-p56 lck complexes that include hematopoietic proteins VAV, SLP-76, and FYB. VAV possesses several domains, including a guanine nucleotide exchange factor (GEF) domain for the Rho and Rac small GTP-binding proteins (14-16). In turn, VAV binds to another hematopoietic protein, , which possesses a C-terminal SH2 domain and proline-rich motifs (20). ZAP-70 is the key kinase that phosphorylates 22), allowing the SH2 domain to bind to two pYESP motifs (Y-113 and Y-128) within the protein (22). VAV and SLP-76 cooperate to augment TcR ͞CD3 induced IL-2 transcription (17, 18). VAV-SLP-76 complex formation, however, is not essential for TcR-induced IL-2 production in all T cells (22). Instead, SLP-76 requires an intact SLP-76 SH2 domain in its pote...

We report here that des-methyl, des-amino pateamine A (DMDA-PatA), a structurally simplified analogue of the marine natural product pateamine A, has potent antiproliferative activity against a wide variety of human cancer cell lines while showing relatively low cytotoxicity against nonproliferating, quiescent human fibroblasts. DMDAPatA retains almost full in vitro potency in P-glycoprotein-overexpressing MES-SA/Dx5-Rx1 human uterine sarcoma cells that are significantly resistant to paclitaxel, suggesting that DMDA-PatA is not a substrate for P-glycoprotein-mediated drug efflux. Treatment of proliferating cells with DMDA-PatA leads to rapid shutdown of DNA synthesis in the S phase of the cell cycle. Cell-free studies show that DMDA-PatA directly inhibits DNA polymerases α and γ in vitro albeit at concentrations considerably higher than those that inhibit cell proliferation. DMDA-PatA shows potent anticancer activity in several human cancer xenograft models in nude mice, including significant regressions observed in the LOX and MDA-MB-435 melanoma models. DMDA-PatA thus represents a promising natural product-based anticancer agent that warrants further investigation.

A high throughput (HT) paradigm generating LC-MS-UV-ELSD based natural product libraries to discover compounds with new bioactivities and or molecular structures is presented. To validate this methodology an extract of the Indo Pacific marine sponge Cacospongia mycofijiensis was evaluated using assays involving cytoskeletal profiling, tumor cell lines, and parasites. Twelve known compounds were identified including the latrunculins (1–4, 10), fijianolides (5, 8–9), mycothiazole (11), the aignopsanes (6–7) and sacrotride A (13). Compounds 1–4, 5, 8–11 exhibited bioactivity not previously reported against the parasite T. brucei, while 11 showed selectivity for lymphoma (U937) tumor cell lines. Four new compounds were also discovered including: aignopsanoic acid B (13), apo latrunculin T (14), 20-methoxy-fijianolide A (15) and aignopsane ketal (16). Compounds 13 and 16 represent important derivatives of the aignopsane class, 14 exhibited inhibition of T. brucei without disrupting microfilament assembly and 15 demonstrated modest microtubule stabilizing effects. The use of removable well plate libraries to avoid false positives from extracts enriched with only 1–2 major metabolites is also discussed. Overall, these results highlight the advantages of applying modern methods in natural products-based research to accelerate the HT discovery of therapeutic leads and or new molecular structures using LC-MS-UV-ELSD based libraries.