Regulation of local GTP availability controls RAC1 activity and cell invasion

Abstract

Physiological changes in GTP levels in live cells have never been considered a regulatory step of RAC1 activation because intracellular GTP concentration (determined by chromatography or mass spectrometry) was shown to be substantially higher than the in vitro RAC1 GTP dissociation constant (RAC1-GTP Kd). Here, by combining genetically encoded GTP biosensors and a RAC1 activity biosensor, we demonstrated that GTP levels fluctuating around RAC1-GTP Kd correlated with changes in RAC1 activity in live cells. Furthermore, RAC1 co-localized in protrusions of invading cells with several guanylate metabolism enzymes, including rate-limiting inosine monophosphate dehydrogenase 2 (IMPDH2), which was partially due to direct RAC1-IMPDH2 interaction. Substitution of endogenous IMPDH2 with IMPDH2 mutants incapable of binding RAC1 did not affect total intracellular GTP levels but suppressed RAC1 activity. Targeting IMPDH2 away from the plasma membrane did not alter total intracellular GTP pools but decreased GTP levels in cell protrusions, RAC1 activity, and cell invasion. These data provide a mechanism of regulation of RAC1 activity by local GTP pools in live cells.

GTP energy dependence of endocytosis and autophagy in the aging brain and Alzheimer’s disease

“…Figure 1 shows preliminary results of GEVAL530 transfection into primary adult neurons from this AD mouse model compared to non-transgenic mouse neurons. As observed by Bianchi-Smiraglia et al [7], we see a non-uniform distribution of GTP in the processes and edges of the soma when measuring free GTP (Fig. 1Aa), with lower free GTP levels in the 3xTg-AD neurons (Fig.…”

“…The role of Rab7 in AD is yet to be clearly elucidated as both early upregulation and late downregulation of Rab7 have been observed affecting neuronal health in both directions. As emphasized by Bianchi-Smiraglia et al [7], total cellular GTP that is measured in homogenates does not allow assessment of free GTP available locally for the Rab GTPases. Free GTP measures have not been reported yet, so their possible role in age and AD-related impairments in endocytosis is unexplored.…”

Rac1 as a Target to Treat Dysfunctions and Cancer of the Bladder

“…In addition, a diversity of effector proteins can modulate Rac1 function, such as the serine-threonine kinases PAK1 (p21-activated kinase 1), MLK-1-3 (mixed-lineage kinases), p70 S6 kinase, CaMKII and many other kinases [ 20 ]. The local availability of GTP in cells plays a role in the control of Rac1 activity [ 34 ]. The expression and subcellular localization of Rac1 is also regulated at the post-transcriptional level via structural modifications, including phosphorylation, ubiquitination, adenylylation, and lipidation (prenylation, geranylgeranylation, palmitoylation) [ 35 ].…”

Local temporal Rac1-GTP nadirs and peaks restrict cell protrusions and retractions

“…9 , right). However, while Fam49 (family of unknown function 49)/CYRI (CYFIP-related Rac interactor)- and ARHGAP39 (Rho GTPase activating protein 39)–mediated Rac1 inhibition can inhibit protrusions and a large number of guanine nucleotide exchange factors, GTPase activating proteins, guanine nucleotide dissociation inhibitors, and the local GTP pool can control Rac1-GTP levels ( 8 , 9 , 41 ), it remains to be investigated how Rac1-GTP levels may be regulated to form local temporal nadirs and peaks during the heights of protrusions and retractions. Besides Rac1-GTP dynamics, other components may also contribute to the restriction of membrane movement.…”