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Rac regulation of transformation, gene expression, and actin organization by multiple, PAK-independent pathways
J K Westwick1, Q T Lambert, G J Clark
1Department of Pharmacology and Lineberger Comprehensive Cancer Center, University of North Carolina School of Medicine, Chapel Hill, 27599-7038, USA.
Abstract:
Rac1 and RhoA are members of the Rho family of Ras-related proteins and function as regulators of actin cytoskeletal organization, gene expression, and cell cycle progression. Constitutive activation of Rac1 and RhoA causes tumorigenic transformation of NIH 3T3 cells, and their functions may be required for full Ras transformation. The effectors by which Rac1 and RhoA mediate these diverse activities, as well as the interrelationship between these events, remain poorly understood. Rac1 is distinct from RhoA in its ability to bind and activate the p65 PAK serine/threonine kinase, to induce lamellipodia and membrane ruffling, and to activate the c-Jun NH2-terminal kinase (JNK). To assess the role of PAK in Rac1 function, we identified effector domain mutants of Rac1 and Rac1-RhoA chimeric proteins that no longer bound PAK. Surprisingly, PAK binding was dispensable for Rac1-induced transformation and lamellipodium formation, as well as activation of JNK, p38, and serum response factor (SRF). However, the ability of Rac1 to bind to and activate PAK correlated with its ability to stimulate transcription from the cyclin D1 promoter. Furthermore, Rac1 activation of JNK or SRF, or induction of lamellipodia, was neither necessary nor sufficient for Rac1 transforming activity. Finally, the signaling pathways that mediate Rac1 activation of SRF or JNK were distinct from those that mediate Rac1 induction of lamellipodia. Taken together, these observations suggest that Rac1 regulates at least four distinct effector-mediated functions and that multiple pathways may contribute to Rac1-induced cellular transformation.
Insights
Rac1 and RhoA are key regulators of cell functions. This study found Rac1
Area of Science:
- Cell Biology
- Molecular Biology
- Oncology
Background:
- Rac1 and RhoA are Rho family proteins regulating actin cytoskeleton, gene expression, and cell cycle.
- Constitutive activation of Rac1/RhoA can lead to cell transformation and tumorigenesis.
- The specific effectors and pathways mediating Rac1/RhoA functions are not fully understood.
Purpose of the Study:
- Investigate the role of p65 PAK (p21-activated kinase) in Rac1-mediated cellular functions.
- Determine the relationship between PAK binding and Rac1-induced transformation, lamellipodium formation, and signaling pathway activation.
- Elucidate the distinct effector pathways contributing to Rac1's diverse cellular activities.
Main Methods:
- Generated effector domain mutants of Rac1 and Rac1-RhoA chimeric proteins that do not bind PAK.
- Assessed Rac1-induced NIH 3T3 cell transformation, lamellipodium formation, and activation of JNK, p38, and SRF.
- Analyzed transcription from the cyclin D1 promoter.
- Investigated signaling pathways mediating Rac1 activation of SRF, JNK, and lamellipodia.
Main Results:
- PAK binding was not essential for Rac1-induced transformation, lamellipodium formation, or activation of JNK, p38, and SRF.
- Rac1's ability to bind and activate PAK correlated with stimulation of cyclin D1 promoter activity.
- Rac1 activation of JNK or SRF, or lamellipodium induction, was neither necessary nor sufficient for transformation.
- Signaling pathways for Rac1 activation of SRF/JNK differed from those for lamellipodia induction.
Conclusions:
- Rac1 regulates at least four distinct effector-mediated functions.
- Multiple pathways contribute to Rac1-induced cellular transformation.
- PAK is involved in specific Rac1 functions, like cyclin D1 transcription, but not others like transformation or lamellipodia formation.