RAC1-dependent regulation of cholinergically induced lamellar protrusive activity is independent of MAPKinase and attenuated by active p-JNK

Abstract

In SH-SY5Y neuroblastoma cell bodies and growth cones the actin dynamics of cholinergically induced lamellar protrusion is demonstrated by live-imaging. Failure of this reaction in cells over-expressing a dominant negative RAC1-mutant (RAC1(T17N)) confirmed that the actin-dynamics of lamellar protrusion is also, in these cells, RAC1-dependent. Pretreatment of untransfected cells with 20 microM UO126 for 2 h, shown to down-regulate the basic level and to completely inhibit cholinergic activation of mitoge-activated kinase (MAPK; erk1/erk2), had no effect neither on spontaneous nor on induced lamellar protrusions. When p-JNK, whose basic activity is very low and not enhanced by a cholinergic stimulation of the cells for up to 30 min, is activated in the presence of 50 microM anisomycin, both spontaneous and cholinergically induced lamellar protrusive activity is attenuated. These data indicate that the RAC1-controlled cascades promoting lamellar protrusion are independent of MAPK activity and partially down-regulated by p-JNK.

Characterization of the smallArabidopsis thalianaGTPase and ADP-ribosylation factor-like 2 protein TITAN 5

“…Third, the mGDP dissociation from TTN5 T30N (k off = 0.149 s -1 ) was 12.5-fold faster than that of TTN5 WT and 37-fold faster than the mGppNHp dissociation of TTN5 T30N (k off = 0.004 s -1 ) (Figure 2D, E; Supplementary Figure S3H, S4G). Mutants of CDC42, RAC1, RHOA, ARF6, RAD, GEM and RAS GTPases, equivalent to TTN5 T30N , display decreased nucleotide binding affinity and therefore tend to remain in a nucleotide-free state in a complex with their cognate GEFs (Erickson et al 1997, Ghosh et al 1999, Radhakrishna et al 1999, Jung and Rösner 2002, Kuemmerle and Zhou 2002, Wittmann et al 2003, Nassar et al 2010, Huang et al 2013, Chang and Colecraft 2015, Fisher et al 2020, Shirazi et al 2020). Since TTN5 T30N exhibits fast guanine nucleotide dissociation, these results suggest that TTN5 T30N may also act in either a dominant-negative or fast-cycling manner as reported for other GTPase mutants (Fiegen et al 2004, Wang et al 2005, Fidyk et al 2006, Klein et al 2006, Soh and Low 2008, Sugawara et al 2019, Aspenström 2020).…”