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Kinase-deficient Pak1 mutants inhibit Ras transformation of Rat-1 fibroblasts
1Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia 19104, USA.
Abstract:
Among the mechanisms by which the Ras oncogene induces cellular transformation, Ras activates the mitogen-activated protein kinase (MAPK or ERK) cascade and a related cascade leading to activation of Jun kinase (JNK or SAPK). JNK is additionally regulated by the Ras-related G proteins Rac and Cdc42. Ras also regulates the actin cytoskeleton through an incompletely elucidated Rac-dependent mechanism. A candidate for the physiological effector for both JNK and actin regulation by Rac and Cdc42 is the serine/threonine kinase Pak (p65pak). We show here that expression of a catalytically inactive mutant Pak, Pak1(R299), inhibits Ras transformation of Rat-1 fibroblasts but not of NIH 3T3 cells. Typically, 90 to 95% fewer transformed colonies were observed in cotransfection assays with Rat-1 cells. Pak1(R299) did not inhibit transformation by the Raf oncogene, indicating that inhibition was specific for Ras. Furthermore, Rat-1 cell lines expressing Pak1(R299) were highly resistant to Ras transformation, while cells expressing wild-type Pak1 were efficiently transformed by Ras. Pak1(L83,L86,R299), a mutant that fails to bind either Rac or Cdc42, also inhibited Ras transformation. Rac and Ras activation of JNK was inhibited by Pak1(R299) but not by Pak1(L83,L86,R299). Ras activation of ERK was inhibited by both Pak1(R299) and Pak1(L83,L86,R299), while neither mutant inhibited Raf activation of ERK. These results suggest that Pak1 interacts with components essential for Ras transformation and that inhibition can be uncoupled from JNK but not ERK signaling.
Insights
The serine/threonine kinase Pak1 is crucial for Ras oncogene-induced cellular transformation. Inhibiting Pak1 disrupts Ras signaling pathways, affecting both Jun kinase (JNK) and extracellular signal-regulated kinase (ERK) activation.
Area of Science:
- Oncology
- Cell Biology
- Molecular Biology
Background:
- The Ras oncogene drives cellular transformation through signaling cascades like MAPK/ERK and JNK.
- Ras also influences the actin cytoskeleton via Rac-dependent pathways.
- The serine/threonine kinase Pak (p65pak) is a potential effector for Rac and Cdc42 in JNK and actin regulation.
Purpose of the Study:
- To investigate the role of Pak1 as a physiological effector in Ras-mediated cellular transformation.
- To determine if Pak1 inhibition affects Ras-induced activation of JNK and ERK signaling pathways.
- To elucidate the specific interactions between Pak1, Ras, Rac, and Cdc42 in cellular transformation.
Main Methods:
- Expression of catalytically inactive Pak1 mutants (Pak1(R299) and Pak1(L83,L86,R299)) in Rat-1 and NIH 3T3 fibroblasts.
- Cotransfection assays to assess inhibition of Ras-induced transformation.
- Analysis of Ras, Rac, and Raf-mediated activation of JNK and ERK signaling pathways.
Main Results:
- Pak1(R299) significantly inhibited Ras transformation of Rat-1 cells but not NIH 3T3 cells.
- Pak1(R299) specifically inhibited Ras transformation, not transformation by the Raf oncogene.
- Pak1(R299) inhibited Ras and Rac activation of JNK, while Pak1(L83,L86,R299) did not.
- Both Pak1 mutants inhibited Ras activation of ERK, but not Raf activation of ERK.
Conclusions:
- Pak1 is essential for Ras oncogene-induced cellular transformation.
- Pak1's role in Ras transformation is partly mediated through JNK signaling.
- Inhibition of Pak1 can uncouple JNK signaling but not ERK signaling from Ras transformation.