Potentiation of Rho-A-mediated Lysophosphatidic Acid Activity by Hyperinsulinemia

Abstract

We have shown previously that insulin promotes phosphorylation and activation of farnesyltransferase and geranylgeranyltransferase (GGTase) II. We have now examined the effect of insulin on geranylgeranyltransferase I in MCF-7 breast cancer cells. Insulin increased GGTase I activity 3-fold and augmented the amounts of geranylgeranylated Rho-A by 18%. Both effects of the insulin were blocked by an inhibitor of GGTase I, GGTI-286. The insulin-induced increases in the amounts of geranylgeranylated Rho-A resulted in potentiation of the Rho-A-mediated effects of lysophosphatidic acid (LPA) on a serum response element-luciferase construct. Preincubation of cells with insulin augmented the LPA-stimulated serum response element-luciferase activation to 12-fold, compared with just 6-fold for LPA alone (p < 0.05). The potentiating effect of insulin was dose-dependent, inhibited by GGTI-286 and not mimicked by insulin-like growth factor-1. We conclude that insulin activates GGTase I, increases the amounts of geranylgeranylated Rho-A protein, and potentiates the Rho-A-dependent nuclear effects of LPA in MCF-7 breast cancer cells. We have shown previously that insulin promotes phosphorylation and activation of farnesyltransferase and geranylgeranyltransferase (GGTase) II. We have now examined the effect of insulin on geranylgeranyltransferase I in MCF-7 breast cancer cells. Insulin increased GGTase I activity 3-fold and augmented the amounts of geranylgeranylated Rho-A by 18%. Both effects of the insulin were blocked by an inhibitor of GGTase I, GGTI-286. The insulin-induced increases in the amounts of geranylgeranylated Rho-A resulted in potentiation of the Rho-A-mediated effects of lysophosphatidic acid (LPA) on a serum response element-luciferase construct. Preincubation of cells with insulin augmented the LPA-stimulated serum response element-luciferase activation to 12-fold, compared with just 6-fold for LPA alone (p < 0.05). The potentiating effect of insulin was dose-dependent, inhibited by GGTI-286 and not mimicked by insulin-like growth factor-1. We conclude that insulin activates GGTase I, increases the amounts of geranylgeranylated Rho-A protein, and potentiates the Rho-A-dependent nuclear effects of LPA in MCF-7 breast cancer cells. farnesyltransferase geranylgeranyltransferase minimal essential medium serum response element lysophosphatidic acid luciferase geranylgeranyltransferase inhibitor-286 insulin-like growth factor polyacrylamide gel electrophoresis cytomegalovirus β-galactosidase Small molecular weight GTPases of the Ras superfamily play an important role in cell proliferation, differentiation, structural organization, and vesicular trafficking (1Barbacid M. Annu. Rev. Biochem. 1987; 56: 779-827Crossref M. Biochem. The of superfamily and by in response to of by to a for activation of GTPases by farnesyltransferase I I and the of a a to the of and Annu. Rev. Biochem. a geranylgeranylated by Ras the of the and have and The of the a geranylgeranylated by GGTase have that insulin promotes the phosphorylation of the of increases and the amounts of in and cells of increased activity of and increased amounts of and GGTase I the of the to the effect of insulin on GGTase I and the of geranylgeranylated not to Rho-A activation of the serum response element by lysophosphatidic acid examined the insulin-induced increases in the amounts of geranylgeranylated Rho-A the activity of LPA in MCF-7 breast cancer cells. cells insulin and a to and the were serum was serum and were Insulin was acid and acid were was were acid was and were and were gel were was the was and the was a The serum response element-luciferase the luciferase the and the were of MCF-7 breast cancer cells were a of inhibitor-286 was was and was a for GGTase and GGTase were I cells were to minimal essential medium and insulin The cells were and for and with insulin for with and The cells were with and a the the was for the of the was with a and for The was in and The of of was by of of cells were to were for in and and with of were for with insulin were previously M. and to was to the of was the with to the of GGTase I and by of phosphorylation were by and amounts of were by of cells were to The cells were and for and with insulin for and and The cells were with and the was to and an and previously Rho-A and Rho-A were the and M. were by by and by with Ras and that of the and M. We the of the in the amounts of The to was not of cells were to were for in and and The cells were for with insulin GGTI-286. of was and the cells were LPA was and the cells were for an were previously M. and to Rho-A was and by gel a and were with acid and to The of and on the was by The of the gel to the and of the were the and the amounts of were by The amounts of were in of to on cells were to the cells were with a a luciferase the and a was a for and The cells were for with insulin in the of GGTI-286 for and with LPA for cells were with insulin for The cells were with and by a a in the activity was on a the and for by β-galactosidase activity by to the luciferase activity by the β-galactosidase were by with < of that insulin activates GGTase I and increases the amounts of geranylgeranylated of insulin in the of the Rho-A-mediated activation of by with insulin and by LPA a in luciferase was by insulin alone LPA alone the of the response to insulin LPA was and a effect in luciferase the of insulin to on a that of alone and of the effects and and The effect of insulin alone was not the the response to insulin a effect with of insulin have previously shown that insulin a of and GGTase in a of cell and M. M. activity in increases in the amounts of and of in response to growth by of growth and growth factor of of with insulin in insulin potentiates the growth growth factor and effects of insulin blocked by an inhibitor of that the effects of insulin by increased activity of insulin promotes increases in the activity of GGTase and the amounts of geranylgeranylated and M. The of the effect of insulin on of to of effect on the the phosphorylation of the of M. and GGTase M. We now that insulin promotes the phosphorylation of the of GGTase I in MCF-7 cells. of the phosphorylation of the of in of activity M. The of insulin to the phosphorylation of the and and not mimicked by growth growth factor effect the of the insulin cells insulin not to insulin in of activation cells with a to in response to insulin a alone in the amounts of effect on the activity of farnesyltransferase The in and that alone a on effect on phosphorylation of the GGTase I potentiation of the nuclear effect of LPA GGTase I the with and effect on phosphorylation to have an effect on GGTase I the of to the effect of LPA with the of effect on of insulin to GGTase I was the of have shown that effect on the phosphorylation of the of and GGTase was blocked by an inhibitor of the M. M. and by with a of Ras M. that insulin activates in a the to the phosphorylation and activation of the growth the to the of insulin and that for insulin to of to GGTase I in response to that insulin GGTase I to We have that insulin increases the activity of GGTase I and the amounts of geranylgeranylated The that the of of insulin Rho-A a of the Ras superfamily of have and have shown to acid to for important in the the the of to and GTPases on the the activity of the of an important that to the effects of LPA on the phosphorylation of nuclear cell M. Biochem. M. and have of and that of to important of the the of Rho-A to cell M. M. and was to for the of cells a cell M. to play an important role in the that M. and play a role to Ras in with and shown to the factor and of and M. The and an of the with a of the Ras in to of with by increased of geranylgeranylated The by promotes the phosphorylation and activation of GGTase I The effect of insulin on GGTase I blocked by the GGTase I GGTI-286 was not to a effect of GGTI-286 shown by the of effect on LPA by of to the of the increases in the of geranylgeranylated to of in response to in nuclear of cell and to in MCF-7 breast cancer cells that insulin increases GGTase I the amounts of and the activity of the effect of insulin on the amounts of Rho-A a in the of Rho-A and 6-fold to of the nuclear effect of LPA nuclear effects of and in M. M. and insulin the amounts of that geranylgeranylated by GGTase a of the effect of insulin a of that the of cancer cells to and that insulin a of M. that not an to the of and The effect of insulin on the and for the of The of insulin to the of growth and an of the of with an increased of and cancer of the and Small molecular weight GTPases of the Ras superfamily play an important role in cell proliferation, differentiation, structural organization, and vesicular trafficking (1Barbacid M. Annu. Rev. Biochem. 1987; 56: 779-827Crossref M. Biochem. The of superfamily and by in response to of by to a for activation of GTPases by farnesyltransferase I I and the of a a to the of and Annu. Rev. Biochem. a geranylgeranylated by Ras the of the and have and The of the a geranylgeranylated by GGTase II. We have that insulin promotes the phosphorylation of the of increases and the amounts of in and cells of increased activity of and increased amounts of and GGTase I the of the to the effect of insulin on GGTase I and the of geranylgeranylated not to Rho-A activation of the serum response element by lysophosphatidic acid examined the insulin-induced increases in the amounts of geranylgeranylated Rho-A the activity of LPA in MCF-7 breast cancer cells. cells insulin and a to and the were serum was serum and were Insulin was acid and acid were was were acid was and were and were gel were was the was and the was a The serum response element-luciferase the luciferase the and the were of MCF-7 breast cancer cells were a of inhibitor-286 was was and was a for GGTase and GGTase were I cells were to minimal essential medium and insulin The cells were and for and with insulin for with and The cells were with and a the the was for the of the was with a and for The was in and The of of was by of of cells were to were for in and and with of were for with insulin were previously M. and to was to the of was the with to the of GGTase I and by of phosphorylation were by and amounts of were by of cells were to The cells were and for and with insulin for and and The cells were with and the was to and an and previously Rho-A and Rho-A were the and M. were by by and by with Ras and that of the and M. We the of the in the amounts of The to was not of cells were to were for in and and The cells were for with insulin GGTI-286. of was and the cells were LPA was and the cells were for an were previously M. and to Rho-A was and by gel a and were with acid and to The of and on the was by The of the gel to the and of the were the and the amounts of were by The amounts of were in of to on cells were to the cells were with a a luciferase the and a was a for and The cells were for with insulin in the of GGTI-286 for and with LPA for cells were with insulin for The cells were with and by a a in the activity was on a the and for by β-galactosidase activity by to the luciferase activity by the β-galactosidase were by with < and the were serum was serum and were Insulin was acid and acid were was were acid was and were and were gel were was the was and the was a The serum response element-luciferase the luciferase the and the were of MCF-7 breast cancer cells were a of inhibitor-286 was was and was a for GGTase and GGTase were GGTase I cells were to minimal essential medium and insulin The cells were and for and with insulin for with and The cells were with and a the the was for the of the was with a and for The was in and The of of was by of of cells were to were for in and and with of were for with insulin were previously M. and to was to the of was the with to the of GGTase I and by of phosphorylation were by and amounts of were by of cells were to The cells were and for and with insulin for and and The cells were with and the was to and an and previously Rho-A and Rho-A were the and M. were by by and by with Ras and that of the and M. We the of the in the amounts of The to was not of cells were to were for in and and The cells were for with insulin GGTI-286. of was and the cells were LPA was and the cells were for an were previously M. and to Rho-A was and by gel a and were with acid and to The of and on the was by The of the gel to the and of the were the and the amounts of were by The amounts of were in of to on cells were to the cells were with a a luciferase the and a was a for and The cells were for with insulin in the of GGTI-286 for and with LPA for cells were with insulin for The cells were with and by a a in the activity was on a the and for by β-galactosidase activity by to the luciferase activity by the β-galactosidase were by with < GGTase I cells were to minimal essential medium and insulin The cells were and for and with insulin for with and The cells were with and a the the was for the of the was with a and for The was in and The of of was by of of cells were to were for in and and with of were for with insulin were previously M. and to was to the of was the with to the of GGTase I and by of phosphorylation were by and amounts of were by of cells were to The cells were and for and with insulin for and and The cells were with and the was to and an and previously Rho-A and Rho-A were the and M. were by by and by with Ras and that of the and M. We the of the in the amounts of The to was not of cells were to were for in and and The cells were for with insulin GGTI-286. of was and the cells were LPA was and the cells were for an were previously M. and to Rho-A was and by gel a and were with acid and to The of and on the was by The of the gel to the and of the were the and the amounts of were by The amounts of were in of to on cells were to the cells were with a a luciferase the and a was a for and The cells were for with insulin in the of GGTI-286 for and with LPA for cells were with insulin for The cells were with and by a a in the activity was on a the and for by β-galactosidase activity by to the luciferase activity by the β-galactosidase were by with < GGTase I cells were to minimal essential medium and insulin The cells were and for and with insulin for with and The cells were with and a the the was for the of the was with a and for The was in and The of of was by MCF-7 cells were to minimal essential medium and insulin The cells were and for and with insulin for with and The cells were with and a the the was for the of the was with a and for The was in and The of of was by of of cells were to were for in and and with of were for with insulin were previously M. and to was to the of was the with to the of GGTase I and by of phosphorylation were by and amounts of were by MCF-7 cells were to were for in and and with of were for with insulin were previously M. and to was to the of was the with to the of GGTase I and by of phosphorylation were by and amounts of were by of cells were to The cells were and for and with insulin for and and The cells were with and the was to and an and previously Rho-A and Rho-A were the and M. were by by and by with Ras and that of the and M. We the of the in the amounts of The to was not MCF-7 cells were to The cells were and for and with insulin for and and The cells were with and the was to and an and previously Rho-A and Rho-A were the and M. were by by and by with Ras and that of the and M. We the of the in the amounts of The to was not of cells were to were for in and and The cells were for with insulin GGTI-286. of was and the cells were LPA was and the cells were for an were previously M. and to Rho-A was and by gel a and were with acid and to The of and on the was by The of the gel to the and of the were the and the amounts of were by The amounts of were in of to MCF-7 cells were to were for in and and The cells were for with insulin GGTI-286. of was and the cells were LPA was and the cells were for an were previously M. and to Rho-A was and by gel a and were with acid and to The of and on the was by The of the gel to the and of the were the and the amounts of were by The amounts of were in of to on cells were to the cells were with a a luciferase the and a was a for and The cells were for with insulin in the of GGTI-286 for and with LPA for cells were with insulin for The cells were with and by a a in the activity was on a the and for by β-galactosidase activity by to the luciferase activity by the β-galactosidase MCF-7 cells were to the cells were with a a luciferase the and a was a for and The cells were for with insulin in the of GGTI-286 for and with LPA for cells were with insulin for The cells were with and by a a in the activity was on a the and for by β-galactosidase activity by to the luciferase activity by the β-galactosidase were by with < were by with < of that insulin activates GGTase I and increases the amounts of geranylgeranylated of insulin in the of the Rho-A-mediated activation of by with insulin and by LPA a in luciferase was by insulin alone LPA alone the of the response to insulin LPA was and a effect in luciferase the of insulin to on a that of alone and of the effects and and The effect of insulin alone was not the the response to insulin a effect with of insulin have previously shown that insulin a of and GGTase in a of cell and M. M. activity in increases in the amounts of and of in response to growth by of growth and growth factor of of with insulin in insulin potentiates the growth growth factor and effects of insulin blocked by an inhibitor of that the effects of insulin by increased activity of insulin promotes increases in the activity of GGTase and the amounts of geranylgeranylated and M. The of the effect of insulin on of to of effect on the the phosphorylation of the of M. and GGTase M. We now that insulin promotes the phosphorylation of the of GGTase I in MCF-7 cells. of the phosphorylation of the of in of activity M. The of insulin to the phosphorylation of the and and not mimicked by growth growth factor effect the of the insulin cells insulin not to insulin in of activation cells with a to in response to insulin a alone in the amounts of effect on the activity of farnesyltransferase The in and that alone a on effect on phosphorylation of the GGTase I potentiation of the nuclear effect of LPA GGTase I the with and effect on phosphorylation to have an effect on GGTase I the of to the effect of LPA with the of effect on of insulin to GGTase I was the of have shown that effect on the phosphorylation of the of and GGTase was blocked by an inhibitor of the M. M. and by with a of Ras M. that insulin activates in a the to the phosphorylation and activation of the growth the to the of insulin and that for insulin to of to GGTase I in response to that insulin GGTase I to We have that insulin increases the activity of GGTase I and the amounts of geranylgeranylated The that the of of insulin Rho-A a of the Ras superfamily of have and have shown to acid to for important in the the the of to and GTPases on the the activity of the of an important that to the effects of LPA on the phosphorylation of nuclear cell M. Biochem. M. and have of and that of to important of the the of Rho-A to cell M. M. and was to for the of cells a cell M. to play an important role in the that M. and play a role to Ras in with and shown to the factor and of and M. The and an of the with a of the Ras in to of with by increased of geranylgeranylated The by promotes the phosphorylation and activation of GGTase I The effect of insulin on GGTase I blocked by the GGTase I GGTI-286 was not to a effect of GGTI-286 shown by the of effect on LPA by of to the of the increases in the of geranylgeranylated to of in response to in nuclear of cell and to in MCF-7 breast cancer cells that insulin increases GGTase I the amounts of and the activity of the effect of insulin on the amounts of Rho-A a in the of Rho-A and 6-fold to of the nuclear effect of LPA nuclear effects of and in M. M. and insulin the amounts of that geranylgeranylated by GGTase a of the effect of insulin a of that the of cancer cells to and that insulin a of M. that not an to the of and The effect of insulin on the and for the of The of insulin to the of growth and an of the of with an increased of and cancer of the and The of that insulin activates GGTase I and increases the amounts of geranylgeranylated of insulin in the of the Rho-A-mediated activation of by with insulin and by LPA a in luciferase was by insulin alone LPA alone the of the response to insulin LPA was and a effect in luciferase the of insulin to on a that of alone and of the effects and and The effect of insulin alone was not the the response to insulin a effect with of insulin We have previously shown that insulin a of and GGTase in a of cell and M. M. activity in increases in the amounts of and of in response to growth by of growth and growth factor of of with insulin in insulin potentiates the growth growth factor and effects of insulin blocked by an inhibitor of that the effects of insulin by increased activity of insulin promotes increases in the activity of GGTase and the amounts of geranylgeranylated and M. The of the effect of insulin on of to The of effect on the the phosphorylation of the of M. and GGTase M. We now that insulin promotes the phosphorylation of the of GGTase I in MCF-7 cells. of the phosphorylation of the of in of activity M. The of insulin to the phosphorylation of the and and not mimicked by growth growth factor effect the of the insulin cells insulin not to insulin in of activation cells with a to in response to insulin a alone in the amounts of effect on the activity of farnesyltransferase The in and that alone a on effect on phosphorylation of the GGTase I potentiation of the nuclear effect of LPA GGTase I the with and effect on phosphorylation to have an effect on GGTase I the of to the effect of LPA with the of effect on of insulin to GGTase I was the of have shown that effect on the phosphorylation of the of and GGTase was blocked by an inhibitor of the M. M. and by with a of Ras M. that insulin activates in a the to the phosphorylation and activation of the growth the to the of insulin and that for insulin to The of to GGTase I in response to that insulin GGTase I to We have that insulin increases the activity of GGTase I and the amounts of geranylgeranylated The that the of of insulin Rho-A a of the Ras superfamily of have and have shown to acid to for important in the the the of to and GTPases on the the activity of the of Rho-A an important that to the effects of LPA on the phosphorylation of nuclear cell M. Biochem. M. and have of and that of to important of the the of Rho-A to cell M. M. and was to for the of cells a cell M. to play an important role in the that M. and play a role to Ras in with and shown to the factor and of and M. The and an of the with a of the Ras in to of with by increased of geranylgeranylated The by promotes the phosphorylation and activation of GGTase I The effect of insulin on GGTase I blocked by the GGTase I GGTI-286 was not to a effect of GGTI-286 shown by the of effect on LPA by of to the of the increases in the of geranylgeranylated to of in response to in nuclear of cell and to in MCF-7 breast cancer cells that insulin increases GGTase I the amounts of and the activity of the effect of insulin on the amounts of Rho-A a in the of Rho-A and 6-fold to of the nuclear effect of LPA nuclear effects of and in M. M. and insulin the amounts of that geranylgeranylated by GGTase a of the effect of insulin a of that the of cancer cells to and that insulin a of M. that not an to the of and The effect of insulin on the and for the of The of insulin to the of growth and an of the of with an increased of and cancer of the and