Phosphorylation and structure-based functional studies reveal a positive and a negative role for the activation loop of the c-Abl tyrosine kinase.

c-Abl is a nuclear and cytoplasmic tyrosine kinase involved in a variety of cellular growth and differentiation processes. In contrast to its oncogenic counterparts, like BCR-Abl, c-Abl is not constitutively tyrosine phosphorylated and its catalytic activity is very low. Here we report tyrosine phosphorylation of endogenous c-Abl and a concomitant increase in catalytic activity. Using Abl -/- cells reconstituted with mutated c-Abl forms, we show that phosphorylation and activity depend on Tyr412 in the activation loop. Tyr412 is also required for stimulation by PDGF or by cotransfection of active Src. Phosphorylation of Tyr412 can occur autocatalytically by a trans-mechanism and cause activation of otherwise inactive c-Abl, suggesting a positive feedback loop on c-Abl activity. In the recent structure of the Abl catalytic domain bound to the STI-571 inhibitor, unphosphorylated Tyr412 in the activation loop points inward and appears to interfere with catalysis. We mutated residues involved in stabilizing this inhibited form of the activation loop and in positioning Tyr412. These mutations resulted in tyrosine phosphorylation and activation of c-Abl, as if relieving c-Abl from inhibition. Tyr412 is therefore necessary both for activity and for regulation of c-Abl, by stabilizing the inactive or the active conformation of the enzyme in a phosphorylation-dependent manner.

Regulation of human c-Abl tyrosine kinase activity in Xenopus oocytes and acceleration of progesterone-induced G2/M transition by oncogenic forms.

Deregulated activity of the Abl protein tyrosine kinase is oncogenic in humans and in animals. The normal cellular form of the enzyme is maintained at a low state of activity by mechanisms that have not yet been entirely elucidated. In particular, little is known about the trans-acting cellular factors involved. We have tested the activity of human c-Abl microinjected into oocytes of Xenopus laevis. In contrast to versions of Abl capable of transforming mammalian cells, which were highly active when introduced into oocytes, the activity of wild type c-Abl was inhibited. Oncogenic forms of Abl efficiently enhanced the ability of Xenopus oocytes to enter M phase following stimulation by progesterone. Abl-enhanced maturation was normal as judged by accumulation of Mos as well as activation of MAP kinase and Cdc2/CyclinB (MPF). Concomitant with maturation and activation of these kinases, Abl became extensively phosphorylated. Altogether, this suggests that an SH3 domain-dependent Abl regulation mechanism similar to the one observed in mammalian cells operates in Xenopus oocytes. Maturation enhancement by microinjection into Xenopus oocytes represents a useful novel assay for analyzing Abl activity. Moreover, the Xenopus oocyte may be a convenient source of trans-acting Abl regulators for biochemical studies.

Stress-wave-induced injury to retinal pigment epithelium cells in vitro.

BACKGROUND AND OBJECTIVE: To determine the survival of in vitro retinal pigment epithelium (RPE) cells subjected to laser-generated stress transients (shock waves) and compare it to that of other cell lines. STUDY DESIGN/MATERIALS AND METHODS: Normal and transformed human retinal pigment epithelium cell lines were used. The cells were imbedded in a gel to prevent motion and cavitation and located in a thin layer at the bottom of a pipette tube closed at one end by a polyimide film. Stress transients were generated by pulsed excimer laser (193 nm and 248 nm wavelength) ablation of the polyimide film. Cell survival, compared to that of unirradiated cells, was assessed by counting surviving cells. The stress was varied from 300 to 740 bars and the number of shock wave pulses applied varied from 5 to 150. RESULTS: Cell survival decreased sharply at the higher stresses but some cells always survived. The lowest survival rate was 50%. Increasing the number of shock wave pulses did not increase cell killing after 20 pulses, demonstrating a saturation effect. In contrast to the transformed cell line, normal cells could not be killed at the highest stress available to us. CONCLUSION: The susceptibility of RPE cells to damage by stress waves varies with cell line. Transformed retinal pigment epithelium cells are more susceptible than normal ones. Saturation of the damage versus number of pulses is observed and a threshold-like behavior for cell killing versus stress is found. Because at least 50% of the cells survived, normal cell growth can serve to replenish damaged cells.

Endogenous regulation of endothelial cell proliferation.

Bovine aortic endothelial cells (BAEC) in culture have the ability to regulate their own proliferation. We have found that a fraction below 100,000 daltons obtained from the media of confluent cultures of BAEC inhibits tritiated thymidine [3H]TdR incorporation as well as their proliferation. The inhibition is dose- and time-dependent; maximum inhibition of [3H]TdR incorporation occurs 8 hr after cells are released from synchronization and the inhibitory fraction is added. Inhibition is evident at concentrations as low as 50 micrograms/ml and reaches a maximum at 600 micrograms/ml. The blockage of [3H]TdR incorporation is reflected in the inhibition of cell proliferation. In the presence of 400 micrograms of endogenous inhibitor per ml of media, added at the time of plating, the average population doubling time increases from 19 to 41 hr. These findings indicate that, in culture, BAEC can regulate their own proliferation by synthesizing an endogenous inhibitor(s) of proliferation.

Surgical removal of vitreous. Its effect on intraocular fibroblast proliferation in the rabbit.

The effect of surgical removal of vitreous on intraocular fibroblast proliferation was studied in an animal model. Forty-eight young adult pigmented rabbits were vitrectomized; four groups of eight animals subsequently received intravitreal injection of tissue-cultured rabbit dermal fibroblasts in doses of 50,000, 100,000, 250,000, and 750,000 cells. As controls, eight vitrectomized rabbits received intravitreal culture medium alone, and the remaining eight vitrectomized rabbits were left uninjected. An additional 40 normal rabbits received the same injections of the same four doses and served as nonvitrectomized controls. All animals were observed for 28 days after injection. The results were dose related and showed that vitrectomy aggravated intraocular fibroblast proliferation in that traction retinal detachment occurred earlier and was more severe in the vitrectomized than in the nonvitrectomized controls.

Intraocular injection of silicone oil for experimental proliferative vitreoretinopathy.

We evaluated the effect of intraocular silicone oil as an internal tamponade on an animal model of proliferative vitreoretinopathy. Lensectomy and vitrectomy were performed on two groups of pigmented rabbits. Twenty-five rabbit eyes then had 1,000 centistokes' viscosity silicone oil injected into the vitreous cavity; 20 control eyes received balanced salt solution. Fourteen days later, each eye was injected with homologous fibroblasts (ten eyes with 100,000 cells, 25 eyes with 250,000 cells, and ten eyes with 750,000 cells). All membranes that developed after the cell injections in both experimental and control eyes produced traction retinal detachment. The character of the membranes formed and the timing of the traction retinal detachment were the same for both groups. In an active phase of experimental proliferation, the internal tamponade of silicone oil is insufficient to keep the retina in place.

The role of cellular proliferation in an experimental model of massive periretinal proliferation.

Transplantation of from 10,000 to 750,000 autologous and homologous dermal fibroblasts into the vitreous cavity of rabbit eyes resulted in identical clinical findings leading to massive periretinal proliferation. A dose-response relationship between the number of cells injected and the extent of retinal traction that developed was established. The second phase of the experiment evaluated the effects of formalin and cobalt irradiation on the fibroblasts' ability to cause traction retinal detachment. Cell proliferation or the attainment of a critical mass of living cells was necessary for the development of traction retinal detachment.