TI - The effects of CD317 knockdown are dependent on Rac and Rho . AB - Activation of the small GTPases Rho and Rac leads to the formation of actin bundles/fibers , although the Rac -induced fibers are also Rho dependent . Therefore , we hypothesized that the actin bundles observed in CD317 knockdown cells might arise as a result of Rac and/or Rho activity and reasoned that if this were the case , transient expression of dominant-negative Rac in the CD317 knockdown cells should abrogate the knockdown phenotype ( ie , the actin bundles observed in the knockdown cells should be lost ) . This is what we observed ; transient expression of a dominant-negative myc-tagged Rac mutant (Rac T17N) inhibits the formation of the characteristic F-actin structures seen in the CD317 knockdown cells ( 7675% of cells expressing Rac T17N had reduced or no actin bundles ; n = 83 ; Fig 3 A ) , whereas transient expression of a dominant-negative cdc42 mutant does not ( 77% of cells expressing dominant-negative cdc42 showed no change in the degree of actin bundling ; n = 23 ; Fig 3 A ) . Furthermore , as would be expected , incubation of CD317 knockdown cells in the presence of 1 uM of the Rho kinase ( ROCK ) inhibitor Y-27632 for 30 min also leads to loss of these structures (Fig 3 A) . Further consequences of Rho activation are the PHOSphorylation on Ser19 of myosin light chain ( MLC ) by ROCK and inactivation of MLC phosphatase ( Tapon and Hall , 1997 ) . Changes in the Ser19 PHOSphorylation status of MLC can thus be used as a biochemical readout of Rho activation . We observed that the amount of Ser19 -PHOSphorylated MLC in CD317 knockdown cells is >10-fold that detected in control cells ( control cells were untransfected Caco-2 cells , lamin A/C knockdown Caco-2 cells or CD317 knockdown Caco-2 cells rescued by expression of rat CD317 ; Fig. 3 B and Fig . S2 A , available at http : . . . . . . /www.jcb.org/cgi/content/full/jcb. lt @@@@@ gt 200804154/DC ) . These biochemical data are consistent with the observed phenotype and implicate CD317 in aspects of the regulation of Rac and Rho function in Caco-2 cells . However , they do not provide direct evidence that there is an increase in the level of active Rac in CD317 knockdown cells . To address this directly , we performed a pull-down assay for active Rac ( see Materials and methods ) using lysate from polarized Caco-2 cells ( control lamin A/C knockdown , CD317 knockdown , and rescued CD317 knockdown ) . This demonstrated an increase in the amount of active Rac in CD317 knockdown cells compared with that in either control or rescued cells , with levels of active Rac being barely detectable in the control and rescued cells ( Fig 3 C and Fig S2 B ) . Thus , there is an increase in the level of active Rac in polarized Caco-2 CD317 knockdown cells .