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The non-catalytic domains of Drosophila katanin regulate its abundance and microtubule-disassembly activity.

Grode KD, Rogers SL - PLoS ONE (2015)

Bottom Line: First, the MIT domain and linker region of Kat60 decrease its abundance by enhancing its proteasome-dependent degradation.The Drosophila katanin regulatory subunit Kat80, which is required to stabilize Kat60 in cells, conversely reduces the proteasome-dependent degradation of Kat60.Second, the MIT domain and linker region of Kat60 augment its microtubule-disassembly activity by enhancing its association with microtubules.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America.

ABSTRACT
Microtubule severing is a biochemical reaction that generates an internal break in a microtubule and regulation of microtubule severing is critical for cellular processes such as ciliogenesis, morphogenesis, and meiosis and mitosis. Katanin is a conserved heterodimeric ATPase that severs and disassembles microtubules, but the molecular determinants for regulation of microtubule severing by katanin remain poorly defined. Here we show that the non-catalytic domains of Drosophila katanin regulate its abundance and activity in living cells. Our data indicate that the microtubule-interacting and trafficking (MIT) domain and adjacent linker region of the Drosophila katanin catalytic subunit Kat60 cooperate to regulate microtubule severing in two distinct ways. First, the MIT domain and linker region of Kat60 decrease its abundance by enhancing its proteasome-dependent degradation. The Drosophila katanin regulatory subunit Kat80, which is required to stabilize Kat60 in cells, conversely reduces the proteasome-dependent degradation of Kat60. Second, the MIT domain and linker region of Kat60 augment its microtubule-disassembly activity by enhancing its association with microtubules. On the basis of our data, we propose that the non-catalytic domains of Drosophila katanin serve as the principal sites of integration of regulatory inputs, thereby controlling its ability to sever and disassemble microtubules.

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Kat60 lacking the MIT domain or the MIT domain and linker region is not detectably degraded in cells.(A-D) Immunoblots of Drosophila S2 cell lysates prepared from cells stably expressing GFP and copper-inducible Kat60 (A), Kat60 and Myc-Kat80 (B), Kat60-ΔMIT (C), or Kat60-AAA (D) that were treated with both Kat60 and Kat80 UTR dsRNA for 7 days total. The cells described in A-D were also treated with 1.0 (A), 1.0 (B), 0.1 (C), or 0.01 mM CuSO4 (D) for 16 hours, washed with S2M, and treated with DMSO (lanes 1–4) or 50 μM MG132 (lanes 5–8) for 0 (lanes 1 and 5), 4 (lanes 2 and 6), 8 (lanes 3 and 7), or 12 hours (lanes 4–8). Molecular weights (in Kd) are shown on the left.
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pone.0123912.g005: Kat60 lacking the MIT domain or the MIT domain and linker region is not detectably degraded in cells.(A-D) Immunoblots of Drosophila S2 cell lysates prepared from cells stably expressing GFP and copper-inducible Kat60 (A), Kat60 and Myc-Kat80 (B), Kat60-ΔMIT (C), or Kat60-AAA (D) that were treated with both Kat60 and Kat80 UTR dsRNA for 7 days total. The cells described in A-D were also treated with 1.0 (A), 1.0 (B), 0.1 (C), or 0.01 mM CuSO4 (D) for 16 hours, washed with S2M, and treated with DMSO (lanes 1–4) or 50 μM MG132 (lanes 5–8) for 0 (lanes 1 and 5), 4 (lanes 2 and 6), 8 (lanes 3 and 7), or 12 hours (lanes 4–8). Molecular weights (in Kd) are shown on the left.

Mentions: Given our unexpected findings that both Kat60-ΔMIT and Kat60-AAA accumulate at higher levels than Kat60 in cells, we next explored how the non-catalytic domains of Drosophila katanin regulate its abundance. Because katanin catalytic subunits from several species have been shown to be ubiquitinated by ubiquitin ligase complexes [33–36] and degraded by the proteasome [36,37], we speculated that the non-catalytic domains of Drosophila katanin regulate its abundance by affecting its degradation via the ubiquitin-proteasome system. To test this hypothesis, we first pulsed the expression of Kat60 alone or together with Myc-Kat80, Kat60-ΔMIT, or Kat60-AAA in cells and then we used immunoblotting to analyze the degradation of these proteins in cells treated with DMSO or the 26S proteasome inhibitor MG132. Whereas Kat60 was markedly degraded in control cells, it was not detectably degraded in cells treated with MG132 (Fig 5A), demonstrating that the degradation of Kat60 is proteasome-dependent. In contrast to Kat60 alone, Kat60 in the presence of Myc-Kat80 was only slightly degraded in control cells (Fig 5B), indicating that Kat80 reduces the proteasome-dependent degradation of Kat60. Strikingly, both Kat60-ΔMIT and Kat60-AAA were not detectably degraded in control cells (Fig 5C and 5D), providing strong evidence that the MIT domain and linker region of Kat60 are required for its proteasome-dependent degradation. From these results, we conclude that the MIT domain and linker region of Kat60 decrease its abundance by enhancing its proteasome-dependent degradation and that Kat80 conversely increases the abundance of Kat60 by reducing its proteasome-dependent degradation.


The non-catalytic domains of Drosophila katanin regulate its abundance and microtubule-disassembly activity.

Grode KD, Rogers SL - PLoS ONE (2015)

Kat60 lacking the MIT domain or the MIT domain and linker region is not detectably degraded in cells.(A-D) Immunoblots of Drosophila S2 cell lysates prepared from cells stably expressing GFP and copper-inducible Kat60 (A), Kat60 and Myc-Kat80 (B), Kat60-ΔMIT (C), or Kat60-AAA (D) that were treated with both Kat60 and Kat80 UTR dsRNA for 7 days total. The cells described in A-D were also treated with 1.0 (A), 1.0 (B), 0.1 (C), or 0.01 mM CuSO4 (D) for 16 hours, washed with S2M, and treated with DMSO (lanes 1–4) or 50 μM MG132 (lanes 5–8) for 0 (lanes 1 and 5), 4 (lanes 2 and 6), 8 (lanes 3 and 7), or 12 hours (lanes 4–8). Molecular weights (in Kd) are shown on the left.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4401518&req=5

pone.0123912.g005: Kat60 lacking the MIT domain or the MIT domain and linker region is not detectably degraded in cells.(A-D) Immunoblots of Drosophila S2 cell lysates prepared from cells stably expressing GFP and copper-inducible Kat60 (A), Kat60 and Myc-Kat80 (B), Kat60-ΔMIT (C), or Kat60-AAA (D) that were treated with both Kat60 and Kat80 UTR dsRNA for 7 days total. The cells described in A-D were also treated with 1.0 (A), 1.0 (B), 0.1 (C), or 0.01 mM CuSO4 (D) for 16 hours, washed with S2M, and treated with DMSO (lanes 1–4) or 50 μM MG132 (lanes 5–8) for 0 (lanes 1 and 5), 4 (lanes 2 and 6), 8 (lanes 3 and 7), or 12 hours (lanes 4–8). Molecular weights (in Kd) are shown on the left.
Mentions: Given our unexpected findings that both Kat60-ΔMIT and Kat60-AAA accumulate at higher levels than Kat60 in cells, we next explored how the non-catalytic domains of Drosophila katanin regulate its abundance. Because katanin catalytic subunits from several species have been shown to be ubiquitinated by ubiquitin ligase complexes [33–36] and degraded by the proteasome [36,37], we speculated that the non-catalytic domains of Drosophila katanin regulate its abundance by affecting its degradation via the ubiquitin-proteasome system. To test this hypothesis, we first pulsed the expression of Kat60 alone or together with Myc-Kat80, Kat60-ΔMIT, or Kat60-AAA in cells and then we used immunoblotting to analyze the degradation of these proteins in cells treated with DMSO or the 26S proteasome inhibitor MG132. Whereas Kat60 was markedly degraded in control cells, it was not detectably degraded in cells treated with MG132 (Fig 5A), demonstrating that the degradation of Kat60 is proteasome-dependent. In contrast to Kat60 alone, Kat60 in the presence of Myc-Kat80 was only slightly degraded in control cells (Fig 5B), indicating that Kat80 reduces the proteasome-dependent degradation of Kat60. Strikingly, both Kat60-ΔMIT and Kat60-AAA were not detectably degraded in control cells (Fig 5C and 5D), providing strong evidence that the MIT domain and linker region of Kat60 are required for its proteasome-dependent degradation. From these results, we conclude that the MIT domain and linker region of Kat60 decrease its abundance by enhancing its proteasome-dependent degradation and that Kat80 conversely increases the abundance of Kat60 by reducing its proteasome-dependent degradation.

Bottom Line: First, the MIT domain and linker region of Kat60 decrease its abundance by enhancing its proteasome-dependent degradation.The Drosophila katanin regulatory subunit Kat80, which is required to stabilize Kat60 in cells, conversely reduces the proteasome-dependent degradation of Kat60.Second, the MIT domain and linker region of Kat60 augment its microtubule-disassembly activity by enhancing its association with microtubules.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America.

ABSTRACT
Microtubule severing is a biochemical reaction that generates an internal break in a microtubule and regulation of microtubule severing is critical for cellular processes such as ciliogenesis, morphogenesis, and meiosis and mitosis. Katanin is a conserved heterodimeric ATPase that severs and disassembles microtubules, but the molecular determinants for regulation of microtubule severing by katanin remain poorly defined. Here we show that the non-catalytic domains of Drosophila katanin regulate its abundance and activity in living cells. Our data indicate that the microtubule-interacting and trafficking (MIT) domain and adjacent linker region of the Drosophila katanin catalytic subunit Kat60 cooperate to regulate microtubule severing in two distinct ways. First, the MIT domain and linker region of Kat60 decrease its abundance by enhancing its proteasome-dependent degradation. The Drosophila katanin regulatory subunit Kat80, which is required to stabilize Kat60 in cells, conversely reduces the proteasome-dependent degradation of Kat60. Second, the MIT domain and linker region of Kat60 augment its microtubule-disassembly activity by enhancing its association with microtubules. On the basis of our data, we propose that the non-catalytic domains of Drosophila katanin serve as the principal sites of integration of regulatory inputs, thereby controlling its ability to sever and disassemble microtubules.

Show MeSH