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CUL3 and protein kinases: insights from PLK1/KLHL22 interaction.

Metzger T, Kleiss C, Sumara I - Cell Cycle (2013)

Bottom Line: We find that kinase activity of PLK1 is redundant for its targeting for CUL3-ubiquitination.Moreover, CUL3/KLHL22 may contact 2 distinct motifs within PLK1 protein, consistent with the bivalent mode of substrate targeting found in other CUL3-based complexes.We discuss these findings in the context of the existing knowledge on other protein kinases and substrates targeted by CUL3-based E3-ligases.

View Article: PubMed Central - PubMed

Affiliation: Institute of Genetics and Molecular and Cellular Biology, Illkirch, France.

ABSTRACT
Posttranslational mechanisms drive fidelity of cellular processes. Phosphorylation and ubiquitination of substrates represent very common, covalent, posttranslational modifications and are often co-regulated. Phosphorylation may play a critical role both by directly regulating E3-ubiquitin ligases and/or by ensuring specificity of the ubiquitination substrate. Importantly, many kinases are not only critical regulatory components of these pathways but also represent themselves the direct ubiquitination substrates. Recent data suggest the role of CUL3-based ligases in both proteolytic and non-proteolytic regulation of protein kinases. Our own recent study identified the mitotic kinase PLK1 as a direct target of the CUL3 E3-ligase complex containing BTB-KELCH adaptor protein KLHL22. (1) In this study, we aim at gaining mechanistic insights into CUL3-mediated regulation of the substrates, in particular protein kinases, by analyzing mechanisms of interaction between KLHL22 and PLK1. We find that kinase activity of PLK1 is redundant for its targeting for CUL3-ubiquitination. Moreover, CUL3/KLHL22 may contact 2 distinct motifs within PLK1 protein, consistent with the bivalent mode of substrate targeting found in other CUL3-based complexes. We discuss these findings in the context of the existing knowledge on other protein kinases and substrates targeted by CUL3-based E3-ligases.

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Figure 2. Catalytic activity of PLK1 is not required for its interaction with KLHL22-adaptor protein. (A) Cells expressing GFP alone and GFP-PLK1 were transfected with HA-KLHL22 and synchronized in mitosis using Taxol or PLK1 inhibitor BI2536. Extracts were immunoprecipitated using GFP-Trap beads. Inputs and immunoprecipitates were analyzed by western blot. (B) Cells expressing GFP alone and GFP-PLK1 only were synchronized and analyzed as in (A). The short (SE) and long (LE) exposures of the representative blots are shown.
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Figure 2: Figure 2. Catalytic activity of PLK1 is not required for its interaction with KLHL22-adaptor protein. (A) Cells expressing GFP alone and GFP-PLK1 were transfected with HA-KLHL22 and synchronized in mitosis using Taxol or PLK1 inhibitor BI2536. Extracts were immunoprecipitated using GFP-Trap beads. Inputs and immunoprecipitates were analyzed by western blot. (B) Cells expressing GFP alone and GFP-PLK1 only were synchronized and analyzed as in (A). The short (SE) and long (LE) exposures of the representative blots are shown.

Mentions: Several substrate-binding mechanisms were identified within the Cullin-RING E3-ligases family. For instance, the SCF E3-ligases require previous phosphorylation of targeted proteins to create a phosphodegron necessary for accurate binding between both components.4,10 In contrast, the APC/C binds to short specific motifs (D-Box, A-box, KEN-box)7 (Fig. 1). The existing data on the regulation of the CUL3 substrate, transcription factor Nrf2, suggest that Nrf2 may not require prior postranslational modification for its binding to CUL3/KEAP113,14 and is constitutively targeteted for ubiquitination under non-stressed cellular conditions. However, little is known about the recognition mechanisms by CUL3-based ligases toward other reported substrates (Fig. 1) in particular protein kinases (Table 1). In order to gain insights into molecular basis of the kinases binding by CUL3, we have tested if the kinase activity of the recently reported substrate PLK11 is required for its binding to CUL3 adaptor KLHL22. For this purpose, GFP tag-protein alone or GFP-tagged PLK1 were expressed in HeLa cells. Subsequently, cells were synchronized in the mitotic stage by addition of Taxol or specific small-molecule inhibitor of PLK1, BI2536,15 and GFP proteins were immunoprecipitated (Fig. 2). As expected, the treatment with BI2536 abolished the phosphorylation-dependent mobility shift of the PLK1 substrate, kinetochore protein BubR116 (Fig. 2A), and reduced amount of the autophosphorylated form of PLK1 (Fig. 2B). In contrast, the HA-tagged KLHL22 (Fig. 2A) and the endogenous KLHL22 (Fig. 2B) efficiently co-immunoprecipitated with PLK1 under these conditions, suggesting that kinase activity of PLK1 is redundant for its recognition by CUL3/KLHL22 E3-ligase. These data are consistent with the fact that strong, salt-resistant interaction was observed in vitro between PLK1 and KLHL22 expressed in bacterial cells, and that downregulation of CUL3/KLHL22 did not modulate kinase activity of PLK1.1 Interestingly, a number of studies suggest that unlike for many SCF E3-ligases,4,10 there is no evidence that posttranslational modifications are required for substrates interactions with CUL3 E3-ligases. Intriguingly in the case of 2 BTB adaptor proteins, KEAP117 and SPOP,18 substrate phosphorylation inhibits rather than promotes their recruitment. Also, regulation of IKKβ by KEAP1 appears to be independent of IKKβ activity,19 and the kinase-dead mutant of PIPKIIβ was more efficiently ubiquitinated, and this CUL3/SPOP-mediated ubiquitination was further enhanced by expression of specific phosphatases.20 A similar situation was observed for DAPK, as the kinase-defective and kinase-active mutants bound KLHL20 as effectively as the wild-type DAPK.21 During mitotic entry, Aurora A kinase is regulated by the CUL3/KLHL18 E3 ligase,22 and this ubiquitination event seems to proceed the activation of Aurora A, suggesting that KLHL18 interacts with an inactive, unmodified kinase. Taken together, all these findings are in a sharp contrast to SCF-mediated mechanisms, in which substrate modification induced by a stimuli switches on the ubiquitination event. One could speculate that for CUL3-mediated ubiquitination events, some specific extra- or intracellular factors generate an “off” signal for substrate targeting. Indeed, Nrf2 is constitutively recognized and ubiquitinated by CUL3/KEAP1, and oxidative stress conditions interfere with E3-ligase activity.13,14 Similarly, DAPK is constitutively targeted to the CUL3-based ligase under basal conditions.21 In both cases, regulation of the substrate ubiquitination occurs at the level of the substrate-specific adaptor rather than substrate itself. While KEAP1 is oxidized on the specific cysteine residues,13,23 KLHL20 is sequestered away from the substrate into PML nuclear bodies under IFN-stress conditions.21 Interestingly, subcellular localization of other mitotic BTB-Kelch proteins involved in CUL3 complexes appears to be regulated in a timely manner during cell cycle progression.1,22,24,25


CUL3 and protein kinases: insights from PLK1/KLHL22 interaction.

Metzger T, Kleiss C, Sumara I - Cell Cycle (2013)

Figure 2. Catalytic activity of PLK1 is not required for its interaction with KLHL22-adaptor protein. (A) Cells expressing GFP alone and GFP-PLK1 were transfected with HA-KLHL22 and synchronized in mitosis using Taxol or PLK1 inhibitor BI2536. Extracts were immunoprecipitated using GFP-Trap beads. Inputs and immunoprecipitates were analyzed by western blot. (B) Cells expressing GFP alone and GFP-PLK1 only were synchronized and analyzed as in (A). The short (SE) and long (LE) exposures of the representative blots are shown.
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Related In: Results  -  Collection

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Figure 2: Figure 2. Catalytic activity of PLK1 is not required for its interaction with KLHL22-adaptor protein. (A) Cells expressing GFP alone and GFP-PLK1 were transfected with HA-KLHL22 and synchronized in mitosis using Taxol or PLK1 inhibitor BI2536. Extracts were immunoprecipitated using GFP-Trap beads. Inputs and immunoprecipitates were analyzed by western blot. (B) Cells expressing GFP alone and GFP-PLK1 only were synchronized and analyzed as in (A). The short (SE) and long (LE) exposures of the representative blots are shown.
Mentions: Several substrate-binding mechanisms were identified within the Cullin-RING E3-ligases family. For instance, the SCF E3-ligases require previous phosphorylation of targeted proteins to create a phosphodegron necessary for accurate binding between both components.4,10 In contrast, the APC/C binds to short specific motifs (D-Box, A-box, KEN-box)7 (Fig. 1). The existing data on the regulation of the CUL3 substrate, transcription factor Nrf2, suggest that Nrf2 may not require prior postranslational modification for its binding to CUL3/KEAP113,14 and is constitutively targeteted for ubiquitination under non-stressed cellular conditions. However, little is known about the recognition mechanisms by CUL3-based ligases toward other reported substrates (Fig. 1) in particular protein kinases (Table 1). In order to gain insights into molecular basis of the kinases binding by CUL3, we have tested if the kinase activity of the recently reported substrate PLK11 is required for its binding to CUL3 adaptor KLHL22. For this purpose, GFP tag-protein alone or GFP-tagged PLK1 were expressed in HeLa cells. Subsequently, cells were synchronized in the mitotic stage by addition of Taxol or specific small-molecule inhibitor of PLK1, BI2536,15 and GFP proteins were immunoprecipitated (Fig. 2). As expected, the treatment with BI2536 abolished the phosphorylation-dependent mobility shift of the PLK1 substrate, kinetochore protein BubR116 (Fig. 2A), and reduced amount of the autophosphorylated form of PLK1 (Fig. 2B). In contrast, the HA-tagged KLHL22 (Fig. 2A) and the endogenous KLHL22 (Fig. 2B) efficiently co-immunoprecipitated with PLK1 under these conditions, suggesting that kinase activity of PLK1 is redundant for its recognition by CUL3/KLHL22 E3-ligase. These data are consistent with the fact that strong, salt-resistant interaction was observed in vitro between PLK1 and KLHL22 expressed in bacterial cells, and that downregulation of CUL3/KLHL22 did not modulate kinase activity of PLK1.1 Interestingly, a number of studies suggest that unlike for many SCF E3-ligases,4,10 there is no evidence that posttranslational modifications are required for substrates interactions with CUL3 E3-ligases. Intriguingly in the case of 2 BTB adaptor proteins, KEAP117 and SPOP,18 substrate phosphorylation inhibits rather than promotes their recruitment. Also, regulation of IKKβ by KEAP1 appears to be independent of IKKβ activity,19 and the kinase-dead mutant of PIPKIIβ was more efficiently ubiquitinated, and this CUL3/SPOP-mediated ubiquitination was further enhanced by expression of specific phosphatases.20 A similar situation was observed for DAPK, as the kinase-defective and kinase-active mutants bound KLHL20 as effectively as the wild-type DAPK.21 During mitotic entry, Aurora A kinase is regulated by the CUL3/KLHL18 E3 ligase,22 and this ubiquitination event seems to proceed the activation of Aurora A, suggesting that KLHL18 interacts with an inactive, unmodified kinase. Taken together, all these findings are in a sharp contrast to SCF-mediated mechanisms, in which substrate modification induced by a stimuli switches on the ubiquitination event. One could speculate that for CUL3-mediated ubiquitination events, some specific extra- or intracellular factors generate an “off” signal for substrate targeting. Indeed, Nrf2 is constitutively recognized and ubiquitinated by CUL3/KEAP1, and oxidative stress conditions interfere with E3-ligase activity.13,14 Similarly, DAPK is constitutively targeted to the CUL3-based ligase under basal conditions.21 In both cases, regulation of the substrate ubiquitination occurs at the level of the substrate-specific adaptor rather than substrate itself. While KEAP1 is oxidized on the specific cysteine residues,13,23 KLHL20 is sequestered away from the substrate into PML nuclear bodies under IFN-stress conditions.21 Interestingly, subcellular localization of other mitotic BTB-Kelch proteins involved in CUL3 complexes appears to be regulated in a timely manner during cell cycle progression.1,22,24,25

Bottom Line: We find that kinase activity of PLK1 is redundant for its targeting for CUL3-ubiquitination.Moreover, CUL3/KLHL22 may contact 2 distinct motifs within PLK1 protein, consistent with the bivalent mode of substrate targeting found in other CUL3-based complexes.We discuss these findings in the context of the existing knowledge on other protein kinases and substrates targeted by CUL3-based E3-ligases.

View Article: PubMed Central - PubMed

Affiliation: Institute of Genetics and Molecular and Cellular Biology, Illkirch, France.

ABSTRACT
Posttranslational mechanisms drive fidelity of cellular processes. Phosphorylation and ubiquitination of substrates represent very common, covalent, posttranslational modifications and are often co-regulated. Phosphorylation may play a critical role both by directly regulating E3-ubiquitin ligases and/or by ensuring specificity of the ubiquitination substrate. Importantly, many kinases are not only critical regulatory components of these pathways but also represent themselves the direct ubiquitination substrates. Recent data suggest the role of CUL3-based ligases in both proteolytic and non-proteolytic regulation of protein kinases. Our own recent study identified the mitotic kinase PLK1 as a direct target of the CUL3 E3-ligase complex containing BTB-KELCH adaptor protein KLHL22. (1) In this study, we aim at gaining mechanistic insights into CUL3-mediated regulation of the substrates, in particular protein kinases, by analyzing mechanisms of interaction between KLHL22 and PLK1. We find that kinase activity of PLK1 is redundant for its targeting for CUL3-ubiquitination. Moreover, CUL3/KLHL22 may contact 2 distinct motifs within PLK1 protein, consistent with the bivalent mode of substrate targeting found in other CUL3-based complexes. We discuss these findings in the context of the existing knowledge on other protein kinases and substrates targeted by CUL3-based E3-ligases.

Show MeSH
Related in: MedlinePlus