Yesterday, I ended a post about FAK, Pyk2 and regulation of RhoA activity by asking “So, what about Rac regulation by [FAK] and Pyk2?”
Today, let’s discuss a paper relating FAK/Pyk2 function studies on Rac1: Regulation of lamellipodial persistence, adhesion turnover, and motility in macrophages by focal adhesion kinase. Katherine Owen, et al., focus on how “Primary bone marrow macrophages isolated from mice in which FAK is conditionally deleted from cells of the myeloid lineage exhibited elevated protrusive activity, altered adhesion dynamics, impaired chemotaxis, elevated basal Rac1 activity, and a marked inability to form stable lamellipodia necessary for directional locomotion.”
Initially, Owen and colleagues found that FAK-null macrophages exhibited lamellipodial protrusions that rapidly formed and retracted, suggesting that these protrusions were unstable, and focal adhesion formation was impaired. This was based on analysis of video microscopy, staining of filamentous actin, kymography, and TIRF microscopy of GFP-Vinculin-expression. Also, they found that the FAK-null macrophages was less capable of chemotaxis towards chemokines CSF-1, SDF-1alpha, and MCP-1, in Boyden chamber experiments, with and without Matrigel.
And they show similar impairment of Matrigel-invasiveness upon siRNA knockdown of Pyk2 in WT and FAK-null macrophages.
The interesting bit for this paper (to me) is when they take a look at Rac1 activity in FAK-null macrophages, as measured by the proportion of GTP-bound Rac1. Owen et al. show an increased basal level of GTP-bound Rac1. (See the immunoblot at the top of this post, from Figure 6A). After reading the paper discussed in yesterday’s post, the lack of mechanistic explanation here is a downer – did they look at any GEFs, for instance? They do however have a portion of the discussion devoted to Rac1 activity in FAK-null cells:
The FAK?/? [FAK-null] macrophages exhibit a similar ruffled and protrusive appearance, which could be reversed by the expression of dominant-negative Rac1-N17 (unpublished data). Thus, the hyperprotrusiveness exhibited in the absence of FAK may be the result of elevated Rac1-GTP. However, Rac1-N17 expression did not rescue the migration defect exhibited by FAK?/? BMMs (unpublished data).
Why didn’t they publish this data? It would’ve been the most interesting part of the paper!
Rac1-N17 would be unresponsive to a guanine nucleotide exchange factor (GEF), essentially generating the same effect as the GEF knockdown experiment described towards the end of yesterday’s post on elevated RhoA activity in FAK-null cells. Further, the phenotype of reduced Rac1 activity would match yesterday’s reduced RhoA activity – restored morphology but not motility. That is, FAK is a vital regulator of two counter-balanced small GTPases required for polarity of focal adhesions.