Proline- and alanine-rich Ste20-related kinase (PASK) is a Ste20-related protein kinase isolated from rat brain. Cell fractionation studies showed that PASK was present both in the cytosol and in Triton X-100-insoluble cytoskeletal fraction in rat tissues. In brain, PASK associated with protein complexes that contained actin and tubulin, confirming the association of PASK with the cytoskeleton in vivo. Glutathione S-transferase-PASK fusion protein cosedimented with F-actin, indicating that PASK binds to F-actin. In contrast to rat tissues, PASK was detected only in the Triton X-100-soluble cytosolic fraction in cultured PC12 and NIH 3T3 cells. Cytosolic PASK translocated to the cytoskeleton when these cells were stimulated with severe cellular stresses such as hypertonic sodium chloride, hydrogen peroxide, and heat shock at 45 degrees C. Our results suggest that PASK may be involved in the regulation of the cytoskeleton in response to cellular stresses such as hyperosmotic shock.