Viral oncogene-induced DNA damage response is activated in Kaposi sarcoma tumorigenesis.

Kaposi sarcoma is a tumor consisting of Kaposi sarcoma herpesvirus (KSHV)-infected tumor cells that express endothelial cell (EC) markers and viral genes like v-cyclin, vFLIP, and LANA. Despite a strong link between KSHV infection and certain neoplasms, de novo virus infection of human primary cells does not readily lead to cellular transformation. We have studied the consequences of expression of v-cyclin in primary and immortalized human dermal microvascular ECs. We show that v-cyclin, which is a homolog of cellular D-type cyclins, induces replicative stress in ECs, which leads to senescence and activation of the DNA damage response. We find that antiproliferative checkpoints are activated upon KSHV infection of ECs, and in early-stage but not late-stage lesions of clinical Kaposi sarcoma specimens. These are some of the first results suggesting that DNA damage checkpoint response also functions as an anticancer barrier in virally induced cancers.

Characterization of the Rab8-specific membrane traffic route linked to protrusion formation.

Rab8 has a drastic effect on cell shape, but the membrane trafficking route it regulates is poorly defined. Here, we show that endogenous and ectopically expressed Rab8 is associated with macropinosomes generated at ruffling membrane domains. These macropinosomes fuse or transform into tubules that move toward the cell center, from where they are recycled back to the leading edge. The biogenesis of these tubules is dependent on actin and microtubular dynamics. Expression of dominant-negative Rab8 mutants or depletion of Rab8 by RNA interference inhibit protrusion formation, but promote cell-cell adhesion and actin stress fiber formation, whereas expression of the constitutively active Rab8-Q67L has the opposite effect. Rab8 localization overlaps with both Rab11 and Arf6, and is functionally linked to Arf6. We also demonstrate that Rab8 activity is needed for the transport of transferrin and the transferrin receptor to the pericentriolar region and to cell protrusions, and that Rab8 controls the traffic of cholera toxin B to the Golgi compartment. Finally, Rab8 colocalizes and binds specifically to a synaptotagmin-like protein (Slp1/JFC1), which is involved in controlling Rab8 membrane dynamics. We propose that Rab8 regulates a membrane-recycling pathway that mediates protrusion formation.

The C-terminal end of R-Ras contains a focal adhesion targeting signal.

R-Ras promotes cell adhesion and activation of integrins through a process that is yet unknown. We show here that active R-Ras (38V) promotes the formation of focal adhesions and a spread cell shape. By contrast, the dominant-negative mutant of R-Ras (43N) reduces the number of focal adhesions, leading to the formation of refractile cells. In adherent cells wild-type R-Ras, activated (38V) R-Ras and endogeous R-Ras were preferentially targeted to focal adhesions, whereas the dominant-negative mutant (43N) of R-Ras was excluded from these structures. Activated mutants of H-Ras and K-Ras were not found in focal adhesions. We dissected R-Ras to find out the determinants that are important for the targeting process. The outermost region in the N-terminus of R-Ras, as well as the intact proline-rich sequence in the C-terminus of RRas that mediates binding to Nck, were not essential. Mutating the potential palmitoylation site (C213A) of RRas results in depalmitoylation and accumulation of R-Ras in the Golgi. Using H-Ras/R-Ras, R-Ras/H-Ras and RRas/K-Ras hybrid molecules we showed that the C-termini (175-218 amino acids) of R-Ras contains the signal for focal adhesions targeting. Exchanging the hypervariable region of H-Ras to R-Ras inhibited the targeting of R-Ras to focal adhesions, whereas H-Ras obtained the ability to localize to focal adhesions after receiving the hypervariable region of R-Ras. This indicates that R-Ras targeting is mediated both by the nucleotide binding status as well as through a specific region in the C-terminus of R-Ras. These results indicate that targeting and activation of R-Ras are linked processes in the formation of focal adhesions.

A Rab8-specific GDP/GTP exchange factor is involved in actin remodeling and polarized membrane transport.

The mechanisms mediating polarized delivery of vesicles to cell surface domains are poorly understood in animal cells. We have previously shown that expression of Rab8 promotes the formation of new cell surface domains through reorganization of actin and microtubules. To unravel the function of Rab8, we used the yeast two-hybrid system to search for potential Rab8-specific activators. We identified a coil-coiled protein (Rabin8), homologous to the rat Rabin3 that stimulated nucleotide exchange on Rab8 but not on Rab3A and Rab5. Furthermore, we show that rat Rabin3 has exchange activity on Rab8 but not on Rab3A, supporting the view that rat Rabin3 is the rat equivalent of human Rabin8. Rabin8 localized to the cortical actin and expression of Rabin8 resulted in remodeling of actin and the formation of polarized cell surface domains. Activation of PKC by phorbol esters enhanced translocation of both Rabin8 and Rab8-specific vesicles to the outer edge of lamellipodial structures. Moreover, coexpression of Rabin8 with dominant negative Rab8 (T22N) redistributes Rabin8 from cortical actin to Rab8-specific vesicles and promotes their polarized transport to cell protrusions. The C-terminal region of Rabin8 plays an essential role in this transport. We propose that Rabin8 is a Rab8-specific activator that is connected to processes that mediate polarized membrane traffic to dynamic cell surface structures.