Localization of p21-activated protein kinase gamma-PAK/Pak2 in the endoplasmic reticulum is required for induction of cytostasis.

The intracellular localization and physiological functions of the p21-activated protein kinase gamma-PAK have been examined in human embryonic kidney 293T and COS-7 cells. At 1-4 days post-transfection, cell division is inhibited by the expression of wild type (WT) gamma-PAK and the mutant S490A, whereas cells expressing S490D and the inactive mutants K278R and T402A grow exponentially, indicating a role for gamma-PAK in the induction of cytostasis. WT gamma-PAK and S490A are localized in a region surrounding the nucleus identified as the endoplasmic reticulum (ER), as determined by immunofluorescence, whereas K278R, T402A, and S490D lack localization. As shown by sucrose density gradient centrifugation, WT gamma-PAK, S490A, and endogenous gamma-PAK are distributed among the high density (ER-associated), intermediate density, and low density fractions, whereas the mutants that do not inhibit cell division are present only as soluble enzyme. The amount of endogenous gamma-PAK associated with the particulate fractions is increased 4-fold when cell division is inhibited by ionizing radiation. gamma-PAK in the ER and intermediate density fractions has high specific activity and is active, whereas the soluble form of gamma-PAK has low activity and is activable. The importance of localization of gamma-PAK is supported by data with the C-terminal mutants S490D and Delta 488; these mutants have high levels of protein kinase activity but do not induce cytostasis and are not bound to the ER. A model for the induction of cytostasis by gamma-PAK through targeting of gamma-PAK to the ER is presented in which gamma-PAK activity and Ser-490 are implicated in the regulation of cytostasis.