Signals from the Ras, Rac, and Rho GTPases converge on the Pak protein kinase in Rat-1 fibroblasts

Abstract

Ras plays a key role in regulating cellular proliferation, differentiation, and transformation. Raf is the major effector of Ras in the Ras > Raf > Mek > extracellular signal-activated kinase (ERK) cascade. A second effector is phosphoinositide 3-OH kinase (PI 3-kinase), which, in turn, activates the small G protein Rac. Rac also has multiple effectors, one of which is the serine threonine kinase Pak (p65(Pak)). Here we show that Ras, but not Raf, activates Pak1 in cotransfection assays of Rat-1 cells but not NIH 3T3 cells. We tested agents that activate or block specific components downstream of Ras and demonstrate a Ras > PI 3-kinase > Rac/Cdc42 > Pak signal. Although these studies suggest that the signal from Ras through PI 3-kinase is sufficient to activate Pak, additional studies suggested that other effectors contribute to Pak activation. RasV12S35 and RasV12G37, two effector mutant proteins which fail to activate PI 3-kinase, did not activate Pak when tested alone but activated Pak when they were cotransfected. Similarly, RacV12H40, an effector mutant that does not bind Pak, and Rho both cooperated with Raf to activate Pak. A dominant negative Rho mutant also inhibited Ras activation of Pak. All combinations of Rac/Raf and Ras/Raf and Rho/Raf effector mutants that transform cells cooperatively stimulated ERK. Cooperation was Pak dependent, since all combinations were inhibited by kinase-deficient Pak mutants in both transformation assays and ERK activation assays. These data suggest that other Ras effectors can collaborate with PI 3-kinase and with each other to activate Pak. Furthermore, the strong correlation between Pak activation and cooperative transformation suggests that Pak activation is necessary, although not sufficient, for cooperative transformation of Rat-1 fibroblasts by Ras, Rac, and Rho.

FIG. 1

Ras and PI 3-kinase activation of Pak in Rat-1 cells. Rat-1 cells were transfected with the indicated plasmids along with a myc-tagged Pak1 construct; extracts were then prepared and used in immune kinase assays. Fold indicates fold activation compared to a vector control (lane 1) as determined by PhosphorImager analysis. MBP, myelin basic protein.

FIG. 2

Effect of dominant negative mutants on Pak activation. (a) Ras activation of Pak is inhibited by dominant negative Rac, Cdc42, and Rho. (b) PI 3-kinase activation of Pak is sensitive to dominant negative Rac and Cdc42 but not dominant negative Rho. MBP, myelin basic protein

FIG. 3

(a) Ras and PI 3-kinase activation of Pak is sensitive to LY294002. Cells were incubated for 90 min with 20 μM LY294002 prior to lysis. (b) Rac activation of Pak is not sensitive to LY294002 (20 μM). (c) Ras and PI 3-kinase dose-response curves for LY294002. MBP, myelin basic protein.

FIG. 4

Pak dominant negative mutants inhibit transformation by Ras effector mutants. Cell transformation was measured by determining growth on soft agar as described in Materials and Methods. (a) Effects of Pak mutants on cell transformation by Ras effector mutants. (b) Effects of Pak mutants on cooperative transformation by Ras effector mutants. (c) Activated Pak cooperates with Raf to transform rv68BUR, a hypersensitive Rat fibroblast cell line. Abbreviations for the Pak mutants: LL, Pak1^L83,L86 (hyperactive Pak1); R, Pak1^R299 (kinase-deficient Pak1); LLR, Pak1^L83,L86,R299 (both kinase-deficient and Rac/Cdc42 binding-deficient Pak1). Ras mutants are abbreviated as S35, G37, and C40, which denote mutations in the effector binding loop of Ras^V12.

FIG. 5

Effects of Pak mutants on Rac/Raf cooperative. (a) Rac/Raf^D340 cooperative transformation is inhibited by Pak dominant negative mutants. (b) Rac/Raf^D340 cooperative activation of ERK is inhibited by Pak dominant negative mutants. (c) Activated Pak cooperates with Raf to stimulate ERK. (d) Pak dominant negative mutants inhibit Pak/Raf^D340 cooperative activation of ERK. MBP, myelin basic protein. Other abbreviations are as in Fig. 4.

FIG. 5

Effects of Pak mutants on Rac/Raf cooperative. (a) Rac/Raf^D340 cooperative transformation is inhibited by Pak dominant negative mutants. (b) Rac/Raf^D340 cooperative activation of ERK is inhibited by Pak dominant negative mutants. (c) Activated Pak cooperates with Raf to stimulate ERK. (d) Pak dominant negative mutants inhibit Pak/Raf^D340 cooperative activation of ERK. MBP, myelin basic protein. Other abbreviations are as in Fig. 4.

FIG. 6

Pak dominant negative mutants inhibit Rho/Raf^D340 cooperation. (a) Transformation assays. (b) ERK kinase assays. MBP, myelin basic protein; HA, hemagglutinin. Other abbreviations are as in Fig. 4.

FIG. 7

Effects of Pak mutants on Rac effector mutants. (a) Pak dominant negative mutants inhibit Rac^V12L37/Raf^D340 cooperative transformation. (b) Pak dominant negative mutants inhibit Rac^V12H40/Raf^D340 cooperative transformation. (c) Rac^V12L37 and Raf^D340 cooperate to activate ERK and are inhibited by Pak dominant negative mutants. (d) Rac^H40 and Raf^D340 cooperate to activate ERK and are inhibited by Pak dominant negative mutants. MBP, myelin basic protein; HA, hemagglutinin. Other abbreviations are as in Fig. 4.

FIG. 8

Cooperative activation of Pak by Ras effector mutants through a PI 3-kinase-independent mechanism. (a) Ras effector mutants cooperate with each other to activate Pak. (b) Ras effector mutants cooperate with Raf to activate Pak. MBP, myelin basic protein. Other abbreviations are as in Fig. 4.

FIG. 9

Activation of Pak by Rac and Rho effector mutants. (a) Rac^V12H40 cooperates with Raf to activate Pak. (b) Rho^V14 cooperates with Raf to activate Pak. L37 is Rac^V12L37, and H40 is Rac^V12H40. MBP, myelin basic protein.

FIG. 10

Model of Ras signaling to Pak and ERK. Dashed lines show possible indirect signals.