Spatiotemporally dynamic microtubule acetylation underlies diverse physiological and pathological events. Despite its ubiquity, the molecular mechanisms that regulate the sole microtubule acetylating agent, α-tubulin-N-acetyltransferase-1 (α-TAT1), remain obscure. Here, we report that dynamic intracellular localization of α-TAT1 along with its catalytic activity determines efficiency of microtubule acetylation. Specifically, we newly identified a conserved signal motif in the intrinsically disordered C-terminus of α-TAT1, consisting of three competing regulatory elements—nuclear export, nuclear import, and cytosolic retention. Their balance is tuned via phosphorylation by CDK1, PKA, and CK2, and dephosphorylation by PP2A. While the unphosphorylated form binds to importins and resides both in cytosol and nucleus, the phosphorylated form binds to specific 14-3-3 adapters and accumulates in the cytosol for maximal substrate access. Unlike other molecules with a similar phospho-regulated signal motif, α-TAT1 uniquely uses the nucleus as a hideout. This allosteric spatial regulation of α-TAT1 function may help uncover a spatiotemporal code of microtubule acetylation in normal and aberrant cell behavior.
Hypoxia is implicated in pathogenesis of many cardiovascular diseases (CVDs), including loss of cardiac contractility; but its effects on cytoskeletal dynamics are not well understood. The microtubule (MT) network provides mechanical strength to cardiomyocytes and its dysregulation is observed in many CVDs. Acetylated MTs provide structural flexibility to cardiomyocytes and protect against proteinopathy-induced cardiac failure. MT acetylation is exclusively catalyzed by α-TAT1, but little is known about how α-TAT1 is regulated. Here we report that hypoxia inhibits MT acetylation and promotes nuclear accumulation of α-TAT1. We show that cytosolic localization of α-TAT1, disrupted in hypoxia, is critical for MT acetylation. Using computational, live cell microscopy and biochemical approaches, we identified a conserved localization motif in the intrinsically disordered C-terminus of α-TAT1, consisting of three competing regulatory elements - nuclear export, nuclear import, and cytosolic retention. Inhibiting Exportin 1 induced nuclear accumulation of α-TAT1. We also found that α-TAT1 cytosolic localization is mediated by CDK1, CK2 and PKA kinases, and inhibited by PP2A phosphatase, through a critical residue: T 322 that binds to 14-3-3 proteins. α-TAT1 knock-out (KO) fibroblasts show defects in actomyosin contractility and focal adhesions. We developed an optogenetic tool, named optoATAT1, which rapidly and reversibly shuttled from nucleus to cytosol on blue light stimulation. optoATAT1 stimulation in HeLa cells increased MT acetylation unlike those kept in dark, validating the tool. optoATAT1 stimulation also led to increase in cell contractility indicated by myosin accumulation and focal adhesion maturation, thus confirming a causal relationship between MT acetylation and cell contractility. Also, α-TAT1 KO fibroblasts are highly proliferative, which may pertain to cardiac hypertrophy. In summary, we have identified a novel role of α-TAT1 C-terminus in regulating MT acetylation and cell contractility under hypoxia. Along with this novel regulatory mechanism of MT acetylation, multiple pharmacological agents identified in this study to modulate spatial regulation of α-TAT1 may offer new insights into the treatment of CVDs.
Spatiotemporal patterns of microtubule modifications such as acetylation underlie diverse cellular functions. While the molecular identity of the acetylating agent, α-tubulin N-acetyltransferase 1 (α-TAT1), as well as the functional consequences of microtubule acetylation have been revealed, the molecular mechanisms that regulate multi-tasking α-TAT1 action for dynamic acetylation remain obscure. Here we identified a signal motif in the intrinsically disordered C-terminus of α-TAT1, which comprises three functional elements - nuclear export, nuclear import and cytosolic retention. Their balance is tuned via phosphorylation by serine-threonine kinases to determine subcellular localization of α-TAT1. While the phosphorylated form binds to 14-3-3 adapters and accumulates in the cytosol for maximal substrate access, the non-phosphorylated form is sequestered inside the nucleus, thus keeping microtubule acetylation minimal. As cancer mutations have been reported to this motif, the unique ensemble regulation of α-TAT1 localization may hint at a role of microtubule acetylation in aberrant physiological conditions.
Microtubules (MTs) provide mechanical strength to cardiomyocytes and dysregulation of MT network is implicated in cardiac diseases. MT acetylation mediates mechanotransduction, provides structural flexibility to cardiomyocytes and protects against proteinopathy-induced cardiac failure. MT acetylation is exclusively catalyzed by α-TAT1, whose only known substrate is α-tubulin in polymerized MTs. However, little is known about how α-TAT1 itself is regulated. Here we report that intracellular spatial localization of α-TAT1 mediates MT acetylation. Specifically, we identified a conserved signal motif in the intrinsically disordered C-terminus of α-TAT1, consisting of three competing regulatory elements - nuclear export, nuclear import and cytosolic retention. Inhibition of Exportin 1-mediated nuclear export induced nuclear accumulation of α-TAT1 and loss of MT acetylation. We found that α-TAT1 nuclear localization is inhibited by CDKs, CK2 and PKA kinases, pharmacological inhibition of which increased nuclear localization of α-TAT1 and inhibited MT acetylation. We identified a critical phosphoThreonine (T 322 ) that binds to 14-3-3 proteins (β, γ, ε and ζ isoforms) downstream of kinases and mediates cytosolic retention of α-TAT1. Inhibition of 14-3-3 proteins also increased nuclear accumulation of α-TAT1. Fibroblastic cells expressing a phosphodeficient α-TAT1 (T322A) show defects in DNA damage response and increased cell proliferation, which may be pertinent to cardiac hypertrophy. Based on these observations, we developed an optogenetic tool, named optoATAT1, which rapidly and reversibly shuttled from the nucleus to the cytosol on blue light stimulation. HeLa cells expressing optoATAT1 exposed to light showed increased MT acetylation unlike those kept in dark, validating the tool. In summary, we have identified a novel role for the C-terminal region of α-TAT1 in regulating its function through dynamic intracellular localization downstream of kinases and 14-3-3 proteins. We have identified multiple pharmacological agents to modulate MT acetylation through spatial regulation of α-TAT1. We have also developed an optogenetic tool to control MT acetylation that will help in elucidating the role of MT acetylation in disease states.
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