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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 4. KLHL22 interacts with 2 domains of PLK1 in cells. Cells expressing GFP alone, GFP fused to full-length PLK1 (GFP-PLK1), kinase domain fragment (GFP-PLK1-KIN), and PBD fragment (GFP-PLK1-PBD) were synchronized in mitosis using Taxol. Extracts were immunoprecipitated using GFP-Trap beads. Inputs and immunoprecipitates were analyzed by western blot. The short (SE) and long (LE) exposures of the representative blots are shown.
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Figure 4: Figure 4. KLHL22 interacts with 2 domains of PLK1 in cells. Cells expressing GFP alone, GFP fused to full-length PLK1 (GFP-PLK1), kinase domain fragment (GFP-PLK1-KIN), and PBD fragment (GFP-PLK1-PBD) were synchronized in mitosis using Taxol. Extracts were immunoprecipitated using GFP-Trap beads. Inputs and immunoprecipitates were analyzed by western blot. The short (SE) and long (LE) exposures of the representative blots are shown.

Mentions: Therefore, we aimed at understanding if a bivalent mode of substrate-CUL3 interaction is also utilized by PLK1. To this end, we expressed and purified the GST-tagged fragments corresponding to entire kinase domain and the regulatory domain, the Polo-box domain (PBD) from bacterial cells. Following incubation with the full-length, bacterially derived MBP-tagged KLHL22 protein, we immunoprecipitated the complexes using GSH-Sepharose. As expected, the full-length PLK1 protein efficiently interacted with the MBP-KLHL22 protein (Fig. 3), consistent with a redundancy for prior posttranslational modifications for this interaction and with the previous findings.1 Interestingly, both PLK1 fragments were able to interact with the full-length KLHL22 protein in vitro (Fig. 3). To corroborate these findings, we expressed the corresponding fragments fused to the GFP-tag in human cells. Following cell synchronization in mitosis by Taxol, we immunoprecipitated the complexes formed. In contrast to the GFP-protein alone, all GFP-fused forms of PLK1 protein were able to efficiently interact with the endogenous KLHL22 protein (Fig. 4). These results suggest that PLK1 utilizes at least 2 distinct binding interfaces located within 2 functional domains of the protein (Fig. 5), consistent with the bivalent model of substrate interaction with the KEAP and SPOP substrate adaptors.17,18,27 While KLHL22 binding to the kinase domain may, in principle, resemble interactions within other BTB-KELCH/kinase complexes, interaction with the PBD domain is likely to be more specific to the family of Polo kinases. The PBD can indeed be only found within PLKs from different species and is critically involved in targeting these kinases to specific subcellular localizations, acting as a phosphoreceptor-biding domain.28-32 It is interesting that the CUL3/KLHL22-mediated ubiquitination takes place at the specific lysine residue (K492) located within the PBD domain and interferes with the phospho-receptor-dependent interactions of PLK1 at the kinetochores.1


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

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

Figure 4. KLHL22 interacts with 2 domains of PLK1 in cells. Cells expressing GFP alone, GFP fused to full-length PLK1 (GFP-PLK1), kinase domain fragment (GFP-PLK1-KIN), and PBD fragment (GFP-PLK1-PBD) were synchronized in mitosis using Taxol. Extracts were immunoprecipitated using GFP-Trap beads. Inputs and immunoprecipitates were analyzed by western blot. The short (SE) and long (LE) exposures of the representative blots are shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3755079&req=5

Figure 4: Figure 4. KLHL22 interacts with 2 domains of PLK1 in cells. Cells expressing GFP alone, GFP fused to full-length PLK1 (GFP-PLK1), kinase domain fragment (GFP-PLK1-KIN), and PBD fragment (GFP-PLK1-PBD) were synchronized in mitosis using Taxol. Extracts were immunoprecipitated using GFP-Trap beads. Inputs and immunoprecipitates were analyzed by western blot. The short (SE) and long (LE) exposures of the representative blots are shown.
Mentions: Therefore, we aimed at understanding if a bivalent mode of substrate-CUL3 interaction is also utilized by PLK1. To this end, we expressed and purified the GST-tagged fragments corresponding to entire kinase domain and the regulatory domain, the Polo-box domain (PBD) from bacterial cells. Following incubation with the full-length, bacterially derived MBP-tagged KLHL22 protein, we immunoprecipitated the complexes using GSH-Sepharose. As expected, the full-length PLK1 protein efficiently interacted with the MBP-KLHL22 protein (Fig. 3), consistent with a redundancy for prior posttranslational modifications for this interaction and with the previous findings.1 Interestingly, both PLK1 fragments were able to interact with the full-length KLHL22 protein in vitro (Fig. 3). To corroborate these findings, we expressed the corresponding fragments fused to the GFP-tag in human cells. Following cell synchronization in mitosis by Taxol, we immunoprecipitated the complexes formed. In contrast to the GFP-protein alone, all GFP-fused forms of PLK1 protein were able to efficiently interact with the endogenous KLHL22 protein (Fig. 4). These results suggest that PLK1 utilizes at least 2 distinct binding interfaces located within 2 functional domains of the protein (Fig. 5), consistent with the bivalent model of substrate interaction with the KEAP and SPOP substrate adaptors.17,18,27 While KLHL22 binding to the kinase domain may, in principle, resemble interactions within other BTB-KELCH/kinase complexes, interaction with the PBD domain is likely to be more specific to the family of Polo kinases. The PBD can indeed be only found within PLKs from different species and is critically involved in targeting these kinases to specific subcellular localizations, acting as a phosphoreceptor-biding domain.28-32 It is interesting that the CUL3/KLHL22-mediated ubiquitination takes place at the specific lysine residue (K492) located within the PBD domain and interferes with the phospho-receptor-dependent interactions of PLK1 at the kinetochores.1

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