A complex containing lysine-acetylated actin inhibits the formin INF2
Posted on: 25 January 2019
Preprint posted on 31 December 2018
Article now published in Nature Cell Biology at http://dx.doi.org/10.1038/s41556-019-0307-4
CAPing INF2 activity: Identification of a cellular factor that facilitates INF2 auto-inhibition
Selected by Laura McCormickCategories: biochemistry, cell biology
Background:
Actin filaments perform an amazingly diverse number of roles in the cell—ranging from cell division to vesicle trafficking to mitochondrial division. Underlying the distinct roles that different populations of polymerized actin can play are the myriad of proteins that regulate actin polymerization.
The formin family of proteins is united by their ability to nucleate actin filaments, overcoming the rate-limiting step of actin polymerization. In recent years, the Higgs lab has extensively studied inverted formin 2 (INF2). In addition to nucleating new actin filaments, INF2 enhances the elongation of actin filaments and accelerates actin depolymerization by severing filaments. Among other roles in the cell, INF2 regulates actin polymerization required for ER-mitochondria calcium transport and mitochondrial division.
A subset of formin proteins are regulated through autoinhibition between the conserved Diaphanous Inhibitory Domain (DID) and the C-terminal Diaphanous Auto-regulatory Domain (DAD). While cellular assays show that regulation of INF2 activity requires the DID-DAD interaction, purified INF2 protein is constitutively active in biochemical assays. To reconcile this disparity, the Higgs lab hypothesized that cellular factor enhances INF2 autoinhibition via the DID-DAD and properly regulates INF2 activity.
Key Findings:
To identify the INF2 inhibitor, the Higgs lab utilized a classic biochemical approach. Mouse brain cytosol was run through various column chromatography steps and fractionated. Each fraction was then mixed with purified INF2 protein to evaluate INF2’s ability to polymerize actin. Fractions that inhibited INF2 activity were pooled and continued through subsequent fractionation until the final Brain Inhibitory Fraction (BIF) was purified. While the BIF inhibited full length INF2 activity, it did not inhibit an INF2 construct lacking the DID domain, suggesting the BIF enhanced INF2 inhibition through DID-DAD interactions.
When the BIF was separated on a SDS-PAGE gel, five protein bands were visualized and sent for mass spectrometry identification. Notably, cyclase associated protein 1 and 2 (CAP1, CAP2) were identified. CAP proteins can bind actin monomers and enhance polymerization, as well as bind actin filaments and enhance severing. While immunodepletion of CAP2 eliminated the strong inhibitory action of the BIF, the authors found recombinant CAP (purified from HEK293 cells) only weakly inhibited INF2 activity in vitro.
Although the differences in inhibitory activity between brain purified and recombinant CAP were puzzling, the authors hypothesized that an addition protein may enhance INF2 inhibition. As CAP binds and co-purifies with actin monomers, they questioned if brain actin vs. HEK293 actin could affect the CAP-INF2 interaction. Following actin-exchange assays, the authors confirmed that recombinant CAP bound to brain actin inhibits INF2 significantly better than CAP bound to HEK293 actin. As most in vitro actin assays utilize actin purified from rabbit or chicken skeletal muscle, the authors also tested the inhibitory action of CAP exchanged with these actin sources. While CAP-chicken actin showed inhibitory activity comparable to CAP-brain actin, CAP-rabbit actin had minimal impact on INF2. To help interpret these disparities in inhibition between actin sources, they probed post-translational modifications of chicken actin by mass spec and identified 4 acetylated residues. Deacetylation of chicken actin by Histone Deacetylase 6 (HDAC6) significantly decreased the inhibitory effect of CAP-chicken actin on INF2 activity, confirming that actin lysine acetylation is key component of CAP-mediated INF2 inhibition.
Fig. 1. TIRF microscopy of actin polymerization in vitro. While full-length INF2 (INF2-FL) alone enhances actin polymerization, the presence of CAP-actin strongly inhibits INF2 activity.
To understand the physiological role of CAP-acetylated actin inhibition and HDAC6-mediated deacetylation, the authors completed several cellular assays. Previous work in several labs, including the Higgs lab, has shown that ionomycin-induced calcium influx in cultured U2OS cells stimulates an INF2-mediated burst of actin polymerization and subsequent mitochondrial division. Interestingly, they found that treatment with Tubastatin A, an inhibitor of HDAC6, blocked the ionomycin-induced actin burst, suggesting that deacetylation of the CAP-actin complex is required for INF2 activation in cells. Supporting this hypothesis, they also found ionomycin treatment transiently decreases the amount of acetylated actin bound to CAP2, as well as the amount of INF2 co-purified with CAP. Interestingly, mutations in the INF2 DID domain have been observed in Charcot-Marie Tooth Disease and Focal Segmental Glomerulosclerosis. Cells transfected with INF2 constructs containing disease-linked mutations exhibit elevated levels of polymerized actin, suggesting these mutations increase basal INF2 activity. Furthermore, these INF2 disease constructs did not co-purify with CAP2 from U2OS cells and were weakly inhibited by CAP-actin in actin pyrene assays. Collectively, these data show that the CAP-acetylated actin complex facilitates INF2 autoinhibition and dysregulation of INF2 activity is linked to disease states.
Fig 2. INF2 auto-inhibition is facilitated by the CAP-acetylated actin complex and is relieved by HDAC-6 mediated deacetylation of actin.
Why I Selected:
- In this paper, the authors employed classic column chromatography—old-school biochemistry—to identify the cellular inhibitor. In the era of proximity-ligation screens such as APEX2 and BioID, reverting to such a traditional method is quite unusual. The amount of the work that went into isolating the BIF is commendable.
- The identification of acetylated actin as a required component of the CAP inhibitory complex is surprising. This paper is the first to show a role for lysine-acetylated actin and it will be interesting to see what other cellular processes lysine-acetylated actin influences.
Questions for the Authors:
- How did the lab begin working on INF2?
- Why did you choose a classic biochemistry approach to identify the INF2 inhibitor?
- Do you hypothesize that other mechanisms exist for relieving INF2 facilitated-autoinhibition besides the deacetylation of actin?
- Interestingly, both INF2 and CAP can contribute to actin polymerization and depolymerization (through severing). Is this purely coincidental or do you think there is a connection there? On a related note, while you’ve shown that CAP-Actin inhibits INF-2 mediated actin polymerization (both nucleation and elongation), does it also inhibit filament severing?
- Do you think acetylated actin may have broader roles than INF2 regulation?
doi: https://doi.org/10.1242/prelights.7849
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