Abstract
The RhoGEF (Rho GTPase guanine-nucleotide-exchange factor) domain of AKAP-Lbc (A-kinase-anchoring protein-Lbc, also known as AKAP13) catalyses nucleotide exchange on RhoA and is involved in the development of cardiac hypertrophy. The RhoGEF activity of AKAP-Lbc has also been implicated in cancer. We have determined the X-ray crystal structure of the complex between RhoA–GDP and the AKAP-Lbc RhoGEF [DH (Dbl-homologous)–PH (pleckstrin homology)] domain to 2.1 Å (1 Å=0.1 nm) resolution. The structure reveals important differences compared with related RhoGEF proteins such as leukaemia-associated RhoGEF. Nucleotide-exchange assays comparing the activity of the DH–PH domain to the DH domain alone showed no role for the PH domain in nucleotide exchange, which is explained by the RhoA–AKAP-Lbc structure. Comparison with a structure of the isolated AKAP-Lbc DH domain revealed a change in conformation of the N-terminal ‘GEF switch’ region upon binding to RhoA. Isothermal titration calorimetry showed that AKAP-Lbc has only micromolar affinity for RhoA, which combined with the presence of potential binding pockets for small molecules on AKAP-Lbc, raises the possibility of targeting AKAP-Lbc with GEF inhibitors.
Highlights
AKAP-Lbc is a member of the A-kinase-anchoring proteins [1,2]
Bacterial overexpression constructs were prepared for RhoA and the DH–pleckstrin homology (PH) domain of AKAP-Lbc (Figure 1A and Table 3)
The RhoGEF activity of AKAP-Lbc is potentially interesting for therapeutic intervention
Summary
AKAP-Lbc ( known as AKAP13) is a member of the A-kinase-anchoring proteins [1,2]. AKAPs are diverse in sequence and domain composition, but have a common feature of binding the PKA (protein kinase A) regulatory subunit and controlling the localization of PKA [3,4,5]. The protein scaffolding mediated by AKAP-Lbc includes KSR1 (kinase suppressor of Ras 1), PKA, RAF, MEK [MAPK (mitogenactivated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase], IKKβ [IκB (inhibitor of nuclear factor κB) kinase β] [6], protein kinases D, C [7,8] and N [9], SHP2 (Src homology 2 domain-containing protein tyrosine phosphatase 2) [10], Hsp (heat-shock protein 20) [11], p38 MAPK and 143-3 proteins [12,13,14]. RhoGEFs activate Rho GTPases allowing extracellular stimuli to promote Rho–GTP formation which initiates signalling pathways inside the cell
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