Requirement of p21-activated Kinase (PAK) for Salmonella typhimurium–induced Nuclear Responses

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Abstract
Salmonella typhimurium has sustained a long-standing association with its host and therefore has evolved sophisticated strategies to multiply and survive within this environment. Central to Salmonella pathogenesis is the function of a dedicated type III secretion system that delivers bacterial effector proteins into the host cell cytoplasm. These effectors stimulate nuclear responses and actin cytoskeleton reorganization leading to the production of proinflammatory cytokines and bacterial internalization. The stimulation of these responses requires the function of Cdc42, a member of the Rho family of small molecular weight GTPases, and SopE, a bacterial effector protein that stimulates guanine nucleotide exchange on Rho GTPases. However, nothing is known about the role of Cdc42 effector proteins in S. typhimurium–induced responses. We showed here that S. typhimurium infection of cultured epithelial cells results in the activation of p21-activated kinase (PAK), a serine/threonine kinase that is an effector of Cdc42-dependent responses. Transient expression of a kinase-defective PAK blocked both S. typhimurium– and SopE-induced c-Jun NH2-terminal kinase (JNK) activation but did not interfere with bacteria-induced actin cytoskeleton rearrangements. Similarly, expression of SH3-binding mutants of PAK did not block actin-mediated S. typhimurium entry into cultured cells. However, expression of an effector loop mutant of Cdc42Hs (Cdc42HsC40) unable to bind PAK and other CRIB (for Cdc42/Rac interacting binding)-containing target proteins resulted in abrogation of both S. typhimurium–induced nuclear and cytoskeletal responses. These results show that PAK kinase activity is required for bacteria-induced nuclear responses but it is not required for cytoskeletal rearrangements, indicating that S. typhimurium stimulates cellular responses through different Cdc42 downstream effector activities. In addition, these results demonstrate that the effector loop of Cdc42 implicated in the binding of PAK and other CRIB-containing target proteins is required for both responses.