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Importance of the field:P21-activated kinases (PAKs) are involved in multiple signal transduction pathways in mammalian cells. PAKs, and PAK1 in particular, play a role in such disorders as cancer, mental retardation and allergy. Cell motility, survival and proliferation, the organization and function of cytoskeleton and extracellular matrix, transcription and translation are among the processes affected by PAK1.Areas covered in this review:We discuss the mechanisms that control PAK1 activity, its involvement in physiological and pathophysiological processes, the benefits and the drawbacks of the current tools to regulate PAK1 activity, the evidence that suggests PAK1 as a therapeutic target and the likely directions of future research.What the reader will gain:The reader will gain a better knowledge and understanding of the areas described above.Take home message:PAK1 is a promising therapeutic target in cancer and allergen-induced disorders. Its suitability as a target in vascular, neurological and infectious diseases remains ambiguous. Further advancement of this field requires progress on such issues as the development of specific and clinically acceptable inhibitors, the choice between targeting one or multiple PAK isoforms, elucidation of the individual roles of PAK1 targets and the mechanisms that may circumvent inhibition of PAK1.

Hyperactivation of NFκB is common in a variety of human diseases. Although numerous transient regulators have been found, it is still unclear which ones can permanently sustain the abnormal status of NFκB in circumvention of several feedback mechanisms and without the associated toxicity. Activation of NFκB could be used to build readouts for genetic screening, and this phenotype has been interrogated using various forward genetics techniques. Mutants with altered activity of NFκB have been obtained using chemical mutagenesis or selected from libraries of full-length cDNAs, genetic suppressor elements or small interfering RNAs. More recently, reversible insertional mutagenesis using retroviral vectors has been tried in this system as well. The authors of the latter report used insertion of a strong promoter to generate dominant mutants, which could be distinguished from the spontaneous mutants by their dependence on the promoter function. In the current issue, the same group describes a similar approach with a new set of tools, based on the modified Sleeping Beauty transposons. Transposon-based insertion of a promoter has been used before for in vivo mutagenesis, but the new study is the first reported use of this strategy in cultured cells. Importantly, the earlier examples of insertional mutagenesis with similar transposons used to produce lists of candidate targets that were chosen among many insertion sites based on certain statistical assumptions or prior knowledge of the nearby genes. In contrast, Dasgupta and colleagues used regulation of the inserted promoter to unambiguously prove the relevance of the targeted locus to the mutant phenotype (Figure 1). The insertional mutant that was characterized in most details revealed expression of the C-terminal fragment of RIP1. The role of RIP1 in signaling to NFκB is well-recognized, yet the finding is unexpected. The N-terminal portion of RIP1 is comprised of a putative kinase domain, although its kinase activity appears dispensable for the activation of NFκB. In the C-terminal portion, the intermediate domain is followed by “RIP homotypic interaction motif” and the DEATH domain, and most confirmed interactors of RIP1 bind to this part of the protein. However, prior reports claimed that a C-terminal fragment produced by caspase cleavage within the intermediate domain of the protein has a dominant-negative effect because in its presence TNFα treatment fails to superinduce an NFκB reporter. Instead, Dasgupta et al. demonstrated that without additional extracellular stimuli a similar short form of RIP1 can activate NFκB and other transcription factors. The state of the cell upon expression of the short RIP1 resembles the consequence of TNFα treatment, including the characteristic dependence on NFκB for survival. However, at the same time the cell is made unresponsive to externally added TNFα because the full-length RIP1, which is the centerpiece of TNFα signaling, is no longer present. This is the most exciting finding of the study: it points to a previously unrecognized mechanism that controls the abundance of RIP1 and, by inference, the responsiveness of the signaling cascades that depend on this molecule. There is now a tantalizing possibility that the same mechanism could be engaged for the therapeutic management of human conditions, such as septic shock, where efficient suppression of TNFα signaling is of vital importance. The presented bioinformatic and experimental datasuggests that a short form of RIP1 may be naturally produced from an internal promoter in the human ripk1 gene. The regulation of this promoter is worth studying further. The longer RIP1 is notoriously toxic, but the shorter variant appears better tolerated. This, and the independence of extracellular stimuli, makes the short RIP1 a more likely candidate for constitutive activation of NFκB in human malignancies. Additional work is needed to confirm this possibility. Further reading:Aggarwal BB. Signalling pathways of the TNF superfamily: a double-edged sword. Nat Rev Immunol 2003; 3:745-56.Dasgupta M, Agarwal MK, Varley P, Lu T, Stark GR, Kandel ES. Transposon-based mutagenesis identifies short RIP1 as an activator of NF-kB. Cell Cycle 2008; 7; In this issue.Kandel ES, Lu T, Wan Y, Agarwal MK, Jackson MW, Stark GR. Mutagenesis by reversible promoter insertion to study the activation of NF-kappaB. Proc Natl Acad Sci USA 2005; 102:6425-30.Kandel ES, Stark GR. "Forward genetics in mammalian cells." Signal Transducers and Activators of Transcription (STATs): Activation and Biology. Ed., Sehgal PB, Levy DE, Hirano T. The Netherlands, Kluwer Academic Publishers.Meylan E, Tschopp J. The RIP kinases: crucial integrators of cellular stress. Trends Biochem Sci 2005; 30:151-9.Perkins ND. Integrating cell-signalling pathways with NF-kappaB and IKK function. Nat Rev Mol Cell Biol 2007; 8:49-62.