InteractiveFly: GeneBrief

Cdc37: Biological Overview | References


Gene name - Cdc37

Synonyms -

Cytological map position - 62B4-62B4

Function - signaling

Keywords - co-chaperone for Hsp83 - tests the proper substrates and establishes stable connection with the client protein to create a Cdc37-client protein binary complex - the binary complex binds to Hsp90 to form a ternary complex - facilitates client protein loading onto the Hsp90 chaperone machinery - maintains the stability of the JNK pathway kinases - physically associates with Insulin receptor to promote neural stem cell reactivation - facilitates signaling in the Wnt and Hedgehog pathways - Aurora B interacts with and requires the Cdc37/Hsp90 complex for its stability

Symbol - Cdc37

FlyBase ID: FBgn0011573

Genetic map position - chr3L:1,795,392-1,797,307

NCBI classification - Cdc37

Cellular location - cytoplasmic



NCBI links: EntrezGene, Nucleotide, Protein
BIOLOGICAL OVERVIEW

Wound closure in the Drosophila larval epidermis mainly involves non-proliferative, endocyling epithelial cells. Consequently, it is largely mediated by cell growth and migration. Both cell growth and migration in Drosophila require the co-chaperone-encoding gene cdc37. Larvae lacking cdc37 in the epidermis failed to close wounds, and the cells of the epidermis failed to change cell shape and polarize. Likewise, wound-induced cell growth was significantly reduced, and correlated with a reduction in the size of the cell nucleus. The c-Jun N-terminal kinase (JNK) pathway, which is essential for wound closure, was not typically activated in injured cdc37 knockdown larvae. In addition, JNK, Hep, Mkk4, and Tak1 protein levels were reduced, consistent with previous reports showing that Cdc37 is important for the stability of various client kinases. Protein levels of the integrin beta subunit and its wound-induced protein expression were also reduced, reflecting the disruption of JNK activation, which is crucial for expression of integrin beta during wound closure. These results are consistent with a role of Cdc37 in maintaining the stability of the JNK pathway kinases, thus mediating cell growth and migration during Drosophila wound healing (Lee, 2019).

The healing of a mammalian skin wound is complex and involves various cellular processes, including blood clotting, inflammation, epithelial cell proliferation and migration, and matrix synthesis and remodeling, which span multiple tissues. In contrast, wound healing in the Drosophila larval epidermis is simple: the epidermis consists mainly of a single, nonproliferative cell layer that underlies the protective cuticle. Thus, wound closure involves primarily cuticle regeneration and cell growth and migration, but not proliferation (Lee, 2019).

Many signaling pathways are required for wound closure in the Drosophila epidermis. Of these, c-Jun N-terminal kinase (JNK), which is required for a broad range of wound healing processes, is the most crucial. Without JNK, cells cannot properly polarize, change shape, orient toward the wound center, or migrate to close the wound. Conversely, some proteins acting upstream of JNK appear to be redundant in a pathway that includes both canonical and noncanonical factors in regard to the embryonic dorsal closure process. Specifically, both JNK and the AP-1 transcription factors DJun (Jra) and DFos (Kay) are absolutely required for wound closure, and larvae that are lacking any one of these factors cannot repair open wounds. In contrast, the Jun/stress-activated protein (SAP) 2 kinases Hep and Mkk4 are partially redundant, as are the Jun/SAP3 kinases Slpr and Tak1 . Although the involvement of the JNK/SAPK pathway in wound healing is well known both in insects and mammals, the mechanisms underlying the regulation of this pathway are not well understood (Lee, 2019).

Protein kinases are often associated with the molecular chaperone Hsp90, which helps these client proteins take on their active conformation. Hsp90 interacts with at least 20 other factors, called cochaperones, which either modulate the activity of Hsp90 or affect the specificity of Hsp90 client proteins. Cdc37 is one such cochaperone that is known to maintain the function and stability of client kinases (Pearl, 2005; Caplan, 2007; Karnitz, 2007; Taipale, 2010), and many kinases are regulated by Cdc37, but the relationship between Cdc37 and the JNK signaling pathway is not clear (Lee, 2019).

A speculative model for the Hsp90-Cdc37-client protein cycle has been suggested (Li, 2018). Cdc37 first tests the proper substrates and establishes stable connection with the client protein to create a Cdc37-client protein binary complex. Then, the binary complex binds to Hsp90 to form a ternary complex. The formation of the Hsp90-Cdc37-client protein ternary complex finally facilitates client protein loading onto the Hsp90 chaperone machinery. It is worthy of note that Cdc37 will be phosphorylated by casein kinase 2 (CK2) at Ser13 before connecting with client proteins and dephosphorylated by the protein phosphatase 5 (PP5) before client protein release (Lee, 2018)

Cdc37 was originally identified as a yeast cell-cycle regulator that was later found to interact with Hsp90 and v-Src (reviewed in Karnitz, 2007). Hsp90 and Cdc37 are both structurally and functionally conserved in metazoans. In Drosophila, cdc37 was initially isolated from a mutagenesis screen based on its involvement in eye development, and was later found to be essential for Sevenless receptor tyrosine kinase signaling (Cutforth, 1994). Null mutations in cdc37 are recessively lethal, indicating it is required for cell viability (Cutforth, 1994; Lange, 2002). Cdc37 inhibits Hh and Wnt signaling pathways in both flies and mammalian cells (Swarup, 2015) and mediates chromosome segregation and cytokinesis by modulating the function of Aurora B kinase (Lee, 2019).

This study isolated cdc37 based on its RNA interference (RNAi) knockdown phenotype in larval epidermal wound closure in Drosophila, and found that cdc37 is required not only for reepithelialization but also for cells to change shape, polarize, and grow during epidermal wound closure, and all of these phenotypes are shared by larvae lacking JNK. Molecularly, cdc37 is required for maintaining the protein levels of JNK pathway components (Lee, 2019).

In Drosophila, JNK mediates diverse wound healing responses, including gene expression, cell shape change and polarization, reepithelialization, and cell fusion. The present study suggests that the JNK pathway also mediates wound-induced endoreplication and cell growth. However, prior reports have indicated that JNK suppresses wound-induced endoreplication in adult stages, which is a discrepancy that requires further investigation (Lee, 2019).

Larvae lacking cdc37 displayed disrupted activation of the JNK pathway and displayed phenotypes similar to those of larvae lacking active JNK. Thus, it is concluded that most of the cdc37-knockdown phenotypes analyzed were likely caused by the disruption of JNK activation during wound healing. It should be noted, however, that cell nucleus size and JNK protein levels were also reduced in the unwounded epidermis of cdc37 knockdown larvae, indicating that loss of cdc37 expression also causes developmental defects. This was not unexpected, given that cdc37 null mutations are cell lethal (Lange, 2002). Considering that A58-GAL4 is only active after early larval stages, and that endoreplicating cells are resistant to apoptosis, the wound healing defects in cdc37-knockdown larvae may have been uncovered luckily due to cell stress caused by wounding in the apoptosis-resistant epidermal cells (Lee, 2019).

Cdc37 is best known as a cochaperone that confers client kinase specificity to Hsp90 (Karnitz, 2007; Taipale, 2010). The client kinases requiring the Hsp90-Cdc37 complex for activity and stability are diverse and include Cdk2, Cdk4, Src, Aurora B, Raf1, and RIP3. However, Cdc37 may also function as an independent molecular chaperone alone, similar to Hsp90. This investigation into the possible involvement of Hsp90 (also known as Hsp83 in Drosophila) in wound healing using multiple Hsp90 RNAi lines did not yield any definitive answer. This study also assessed whether aurora B, cdc2, or ckII were involved in wound healing, as these factors reportedly interact with cdc37 in various contexts. Larvae deficient of each of these factors closed wounds normally. Finally, no noticeable changes were found in the protein level or localization of Cdc37 during wound healing. Thus, the requirement of cdc37 for JNK activation is a novel finding. Nonetheless, defining the detailed molecular mechanisms underlying Cdc37 functions requires further investigation (Lee, 2019).

Hsp83/Hsp90 physically associates with Insulin receptor to promote neural stem cell reactivation

Neural stem cells (NSCs) have the ability to exit quiescence and reactivate in response to physiological stimuli. In the Drosophila brain, insulin receptor (InR)/phosphatidylinositol 3-kinase (PI3K)/Akt pathway triggers NSC reactivation. However, intrinsic mechanisms that control the InR/PI3K/Akt pathway during reactivation remain unknown. This study identified heat shock protein 83 (Hsp83/Hsp90), a molecular chaperone, as an intrinsic regulator of NSC reactivation. Hsp83 is both necessary and sufficient for NSC reactivation by promoting the activation of InR pathway in larval brains in the presence of dietary amino acids. Both Hsp83 and its co-chaperone Cdc37 physically associate with InR. Finally, reactivation defects observed in brains depleted of hsp83 were rescued by over-activation of the InR/PI3K/Akt pathway, suggesting that Hsp83 functions upstream of the InR/PI3K/Akt pathway during NSC reactivation. Given the conservation of Hsp83 and the InR pathway, this finding may provide insights into the molecular mechanisms underlying mammalian NSC reactivation (Huang, 2018).

How the InR/PI3K/Akt pathway is regulated during NSC reactivation is poorly understood. This study shows that molecular chaperone Hsp83/Hsp90, together with its co-chaperone Cdc37, play a role in the reactivation of Drosophila NSCs. Mechanistically, Hsp83 and Cdc37 physically associate with InR and are important for the activation of the InR/PI3K/Akt pathway in NSCs. Therefore, this study demonstrates that Hsp83 serves as an intrinsic factor within NSCs that is necessary for the activation of the InR/PI3K/Akt pathway and, in turn, reactivation of NSCs. This evidence suggests that Hsp83 and Cdc37 regulate the protein folding and activation of InR in the nervous system (Huang, 2018).

The role of Hsp83 in NSC reactivation at early larval stages is distinct from its known role in centrosomes or NSC polarity. Drosophila Hsp83 is a core centrosomal component required for proper mitotic spindle formation and chromosome segregation. In Drosophila larval CNS, Hsp83 and co-chaperone Sgt1 are required for the stabilization of Polo and centrosome organization in NSCs (Martins, 2009). Hsp83 and Sgt1 are also required for the establishment of NSC polarity via the LKB1/AMPK pathway in third-instar larvae. However, sgt1 RNAi in NSCs did not display any phenotypes during NSC reactivation, suggesting that Hsp83 interacts with different co-chaperones to control NSC reactivation and cortical polarity at different developmental stages. Consistent with this notion, Cdc37, but not other co-chaperones of Hsp83, was found to be required for NSC reactivation. The proliferation of MB NSCs were unaffected by hsp83 knockdown, Therefore, Hsp83 promotes NSC reactivation rather than general cell proliferation. Consistent with these observations, there is no significant difference in proliferation between hsp83 mutant and wild-type eye imaginal discs in the proliferating zone. Interestingly, in pupal eyes that undergo terminal differentiation, Hsp83 is required for cell-cycle exit by activating the anaphase-promoting complex/cyclosome. This study found cytokinesis defects in cdc37-depleted NSCs, but not in NSCs depleted of Hsp83. This observation is consistent with a known role of Cdc37 in cell division and cytokinesis in Drosophila (Huang, 2018).

Hsp90 plays a key role in signal transduction and appears to bind to its substrates in a near native state poised for activation by binding of ligand or other factors. Since Hsp83 overexpression is sufficient to drive the activation of InR/PI3K/Akt pathway and trigger premature NSC reactivation, Hsp83 likely plays an active role in promoting NSC reactivation by binding to InR at a late stage of folding poised for activation by dILP binding. Furthermore, in the absence of dietary amino acids, the expression of hsp83 is downregulated, likely partially contributing to the inactivation of the InR pathway. It is proposed that InR is a target of Hsp83 and Cdc37 during NSC reactivation. The physical association among Hsp83, Cdc37, and InR was strongly supported by PLA assays, and both in vitro and in vivo BiFC. Although tandem affinity purification-mass spectrometry in Drosophila S2 cells implied an interaction between Hsp83 and InR, this study failed to detect a consistent interaction between Hsp83 and InRintra in S2 cells in co-immunoprecipitation experiments, probably due to the transient nature of this interaction. In addition, genetic interaction experiments indicate that Hsp83 activates the InR/PI3K/Akt pathway to promote NSC reactivation. Taken together, InR is likely a client of Hsp83 in Drosophila NSCs. Consistent with these findings, in human fibroblasts, Hsp90 co-immunoprecipitated with intracellular InR β. Furthermore, Hsp90 facilitates the maturation of the InR precursor in the ER and, in turn, is required for cell surface expression of InR in both bovine adrenal medullary chromaffin cells and human kidney HEK293 cells. Therefore, the interaction between the Hsp90 chaperone family and InR may be conserved from Drosophila to humans. In mammals, the expression level of Hsp90 in the brain is the highest among all tissues. Although mammalian Hsp90 proteins are heavily implicated in neurodegenerative diseases, their function in brain development is not well understood. Hsp90/Cdc37 stabilize the intracellular domain of Ryk, a Wnt receptor required for neurogenesis (Lyu, 2009). Furthermore, Hsp90 stabilizes hypoxia-inducible factor-1, which promotes NSC proliferation under hypoxia (Xiong, 2009). It remains to be determined whether the interaction between mammalian Hsp90 and InR is conserved during mammalian NSC development (Huang, 2018).

Genome-wide identification of phospho-regulators of Wnt signaling in Drosophila

Evolutionarily conserved intercellular signaling pathways regulate embryonic development and adult tissue homeostasis in metazoans. The precise control of the state and amplitude of signaling pathways is achieved in part through the kinase- and phosphatase-mediated reversible phosphorylation of proteins. In this study, a genome-wide in vivo RNAi screen was performed for kinases and phosphatases that regulate the Wnt pathway under physiological conditions in the Drosophila wing disc. These analyses have identified 54 high-confidence kinases and phosphatases capable of modulating the Wnt pathway, including 22 novel regulators. These candidates were also assayed for a role in the Notch pathway, and numerous phospho-regulators were identified. Additionally, each regulator of the Wnt pathway was evaluated in the wing disc for its ability to affect the mechanistically similar Hedgehog pathway. 29 dual regulators were identified that have the same effect on the Wnt and Hedgehog pathways. As proof of principle, this study established that Cdc37 and Gilgamesh/CK1gamma inhibit and promote signaling, respectively, by functioning at analogous levels of these pathways in both Drosophila and mammalian cells. The Wnt and Hedgehog pathways function in tandem in multiple developmental contexts, and the identification of several shared phospho-regulators serve as potential nodes of control under conditions of aberrant signaling and disease (Swarup, 2015).

Divergent disease states have been attributed to be a cause or consequence of aberrant protein phosphorylation. Wnt signaling is phosphor-regulated both in its silent and active states, but thus far understanding of kinases, phosphatases and associated factors of the pathway has been limited. In this study, the first genome-wide in vivo screen was performed under physiological conditions in the Drosophila wing disc for phospho-regulators of the Wnt pathway. 54 high-confidence regulators were identified, 22 of which are novel. The results of these analyses do not indicate whether a high-confidence regulator has a direct or indirect effect on signaling. However, as ~60% of the high-confidence regulators identified have been previously validated to have a direct effect on Wnt signaling, it is predictd that at least some of the novel high-confidence regulators identified would also have a direct effect on the pathway. Indeed, subsequent analyses of Myopic revealed a novel role in regulating Wg secretion. Although the mechanism and components of the Wnt pathway are for the most part conserved between Drosophila and humans, there are possibly vertebrate-specific phospho-regulators of signaling that would not have been identified in these analyses. The dataset represents the largest list of putative phospho-regulators of the Wnt pathway identified to date, almost all of which have identified human orthologs and are therefore likely to be functionally conserved (Swarup, 2015).

As part of this study, previously unknown relationships were established between the Wnt and Hh pathways in vivo by identifying 12 novel dual regulators that are proposed to function at analogous levels of signaling. As proof of concept, the roles of Cdc37 and Gish/CK1γ were biochemically characterized to demonstrate that their functions are conserved from Drosophila to mammalian cells. An initial analysis is reported of candidate regulators of Notch signaling during wing disc development. Although these findings are preliminary, they highlight an emerging theme of phospho-regulation of Notch that likely hold parallels in vertebrate biology. The comparison of signaling pathways in vivo and the identification of specific versus shared phospho-regulators facilitate understanding of human development and disease states (Swarup, 2015).

Regulation of Greatwall kinase by protein stabilization and nuclear localization

Greatwall (Gwl) functions as an essential mitotic kinase by antagonizing protein phosphatase 2A. This study identified Hsp90, Cdc37 and members of the importin alpha and beta families as the major binding partners of Gwl. Both Hsp90/Cdc37 chaperone and importin complexes associated with the N-terminal kinase domain of Gwl, whereas an intact glycine-rich loop at the N-terminus of Gwl was essential for binding of Hsp90/Cdc37 but not importins. Hsp90 inhibition led to destabilization of Gwl, a mechanism that may partially contribute to the emerging role of Hsp90 in cell cycle progression and the anti-proliferative potential of Hsp90 inhibition. Moreover, in agreement with its importin association, Gwl exhibited nuclear localization in interphase Xenopus S3 cells, and dynamic nucleocytoplasmic distribution during mitosis. KR456/457 was identified as the locus of importin binding and the functional NLS of Gwl. Mutation of this site resulted in exclusion of Gwl from the nucleus. Finally, this study showed that the Gwl nuclear localization is indispensable for the biochemical function of Gwl in promoting mitotic entry (Yamamoto, 2014). Entry into mitosis requires activation of maturation-promoting factor (MPF), a complex of cyclin B and Cdk1. During mitotic entry, MPF activation is triggered by Cdc25-mediated dephosphorylation of Cdk1. In addition to mechanisms that directly control the activity of Cdc25 and MPF, dynamic and regulated nucleocytoplasmic localization of these factors was also demonstrated to be important. In particular, both cyclin B and Cdc25 need to localize to the nucleus to trigger mitotic entry. It has been shown that phosphorylation of cyclin B by Polo-like kinase 1 (Plk1) promotes its nuclear localization. Furthermore, checkpoint kinases, Chk1 and Chk2, phosphorylate Cdc25, and thereby creating a binding site for 14-3-3 proteins which sequestrate Cdc25 in cytoplasm and inhibit M-phase entry. In addition to MPF, another enzyme required for both mitotic entry and maintenance is the Greatwall kinase (Gwl). Recent evidence revealed that Gwl inhibits the activity of protein phosphatase 2A/B55δ, the principal phosphatase acting on Cdk-phosphorylated substrates (Yamamoto, 2014).

The crucial nature of this inhibition was demonstrated by the failure of M-phase maintenance in egg extracts with full activity of MPF and other mitotic kinases but deficient of Gwl function. On the other hand, the presence of Gwl greatly reduced the amount of MPF required for mitotic entry (Yamamoto, 2014).

The mechanism of PP2A/B55δ inhibition by Gwl has been nicely solved with the recent identification of Endosulfine and its related family member, cAMP-regulated phosphoprotein 19 kDa, as substrates of Gwl. These substrates specifically bind to and inhibit PP2A/B55δ when they are phosphorylated by Gwl. While compelling evidence obtained in a variety of experimental systems revealed an essential role of Gwl in mitotic regulation, very little has been learned about how Gwl itself is regulated. It has been shown that Gwl is phosphorylated in M-phase, in quantitative correlation with its kinase activity (Yamamoto, 2014).

Mitotic phosphorylation of Gwl by MPF within its presumptive activation loop is required for Gwl activation, suggesting that MPF acts upstream of Gwl during mitotic entry. It has also been shown that Gwl can be phosphorylated by Plk1, whereas the specific function and regulation of this modification remains to be further clarified (Yamamoto, 2014).

In the search for new regulators of Gwl, a proteomic analysis revealed 2 groups of proteins as the major binding partners of Gwl which were co-purified with Gwl from G2 and M phase Xenopus oocytes. Specifically, Gwl bound both the chaperone complex Hsp90/Cdc37 and importin α/β through its N-terminus. Hsp90 was required to stabilize Gwl in both interphase and M-phase extracts. Furthermore, this study identified the functional NLS in Gwl that mediated its binding to importins. Mutation of amino acid residues KR456/457 within the NLS led to exclusively cytoplasmic localization of the protein in Xenopus S3 cells and interfered its function in promoting mitotic entry in egg extracts (Yamamoto, 2014).

Several heat shock proteins function as molecular chaperones. In particular, Hsp90 has been related to regulators of the cell cycle, including Cdk1, p53, Cdk4, and Plk1. It has been shown that the evolutionarily conserved protein association between Plk1 and Hsp90 was required for the stability of Plk1, and thereby involved in centrosomal functions and metaphase-to-anaphase transition (Yamamoto, 2014).

This study discovered Hsp90 as a major associated partner for Gwl. Further analysis showed that the N-terminal segment of Gwl mediated its association with Hsp90. Interestingly, a glycine-rich loop in the N-terminal Gwl was required for Hsp90 association, and a G41S mutant form of Gwl was deficient in Hsp90 association. Proteomic identification of Gwl-associated proteins also revealed its association with Cdc37, the co-chaperone of Hsp90 involved in recognition of some substrates. Initially identified in budding yeast as a cell division cycle regulator, Cdc37 has been shown to interact with Cdk4, Cdk5, and several other cell cycle factors. Consistently, this study found that G41S Gwl lost the association with Cdc37, confirming the involvement of the glycine-rich loop. Importantly, inhibition of Hsp90 reduced the protein stability of Gwl and suppressed M-phase entry, suggesting that complexing to the Hsp90/Cdc37 chaperone is essential for Gwl function. The study therefore revealed Gwl as a key client protein through which Hsp90/Cdc37 chaperone regulates cell division (Yamamoto, 2014).

This new discovery contributes to better understanding of cell cycle regulation, and yields potential implication to cancer therapy. In fact, Hsp90 is frequently overexpressed in cancer cells, whereas its inhibition exhibits anti-proliferative potentials and is being explored in multiple clinical trials for treatment of various types of cancer. The proper function of cellular enzymes often involves sophisticated regulation of subcellular localization, in addition to enzymatic activity and protein stability. For instance, cyclin B/Cdk1 is imported into the nucleus before nuclear membrane breaks down to promote mitotic entry. Similarly, dynamic subcellular localization of Plk1 and Aurora A has been shown to regulate their functions during mitotic progression. One mechanism to regulate the nuclear localization of a protein is through association with the importin family members. Importin is typically composed of 2 subunits, importin α and importin β. For nuclear transport, importin β either directly binds and transports cargo proteins, or is adapted to the cargo proteins through importin α. This study reports that Gwl associates with both importin α and Importin β, suggesting that the nuclear localization of Gwl is mediated by importin. In cultured Xenopus cells, this study showed that Gwl localized to the nucleus in interphase, and was diffused into the cytoplasm in mitosis. This detailed analysis identified a functional NLS around KR456/457. Mutation of the NLS abolished the association of Gwl with importin α and Importin β, and led to Gwl nuclear exclusion and cytoplasmic sequestration (Yamamoto, 2014).

Independent studies in Drosophila and mammalian cells also characterized the nuclear localization of Gwl. The Drosophila homolog of Gwl contains 2 essential NLS motifs within the central region, mutation of which disrupted its nuclear location. However, neither of these NLS motifs in Drosophila is conserved in vertebrates. The NLS of human Gwl was found around KR444/445, the corresponding residues of KR456/457 in Xenopus Gwl. Thus, the NLS of Xenopus Gwl, as identified in this study, represents a well-conserved mechanism in vertebrate animals (Yamamoto, 2014).

Studies in Drosophila and human cells suggested a pre-mitotic transport of Gwl from nucleus to cytoplasm. It was also observed in Xenopus cells that nuclear Gwl started to diffuse into cytoplasm before centrosomes reached opposite positions. While it remained unclear how this nucleocytoplasmic translocation of Gwl may contribute to the regulation of mitotic entry, previous studies indicated that the translocation was dependent on Gwl phosphorylation by CDK1, and possibly also Plk1 (Yamamoto, 2014).

It was also suggested that the kinase activity of Gwl was required for its nuclear export as a kinase-dead form of Gwl harboring a mutation at the ATP-binding site was deficient in nuclear export. However, a different form of kinase-dead Gwl used in this study exhibited a similar pattern of nucleocytoplasmic translocation as the WT. Notably, this mutant form of Gwl was efficiently phosphorylated in mitosis, as judged by the retarded gel migration. Furthermore, it was observed that Gwl was reconcentrated into the daughter nuclei in telophase, seemingly before mitotic chromosomes are fully decondensed. By comparison, the NLS-deficient form of Gwl remained diffused in the cytoplasm during mitotic exit (Yamamoto, 2014).

An important lesson learned from previous studies on Cdk1/cyclin B, Plk1, Aurora kinases, and other mitotic regulators was that their subcellular localization often constitutes an essential mechanism to ensure the proper function of these factors. Regulation of Gwl nuclear localization is functionally important as the NLS-deficient Gwl was unable to fully rescue the mitotic defects caused by Gwl-depletion in both Drosophila and mammalian cells (Yamamoto, 2014).

To shed new light on the role of Gwl nuclear localization, whether the NLS is required for the biochemical function of Gwl was investigated. It has been shown that Gwl promotes mitotic entry through phosphorylation of Ensa/Arpp-19, which subsequently inhibit PP2A/B55δ. Interestingly, the results showed that the NLS-deficient Gwl failed to promote mitotic entry in extracts supplemented with sperm nuclei, despite that this mutant possesses intact and functional kinase domains and promoted mitotic entry in extracts devoid of nucleus formation. Notably, a number of previous studies also characterized the nuclear import of cyclin B1 and Cdc25C in Xenopus egg extracts (Yamamoto, 2014).

These studies argue that even though Xenopus egg extracts can biochemically undergo cell cycle progression in the absence of nuclei, nucleocytoplasmic regulation of the cell cycle is recapitulated in this system when sperm nuclei are reconstituted. As another example, although Xenopus egg extracts support DNA replication without nuclei, it has been shown that the NLS of cyclin E is required for DNA replication in Xenopus egg extracts with the supplementation of sperm nuclei. Moreover, the nuclear import of cyclin B1 is required for mitotic entry in Xenopus oocytes, despite that enucleated Xenopus oocytes still exhibit mitotic activation of Cdk1 (Yamamoto, 2014).

While future analyses are required to reveal how the nuclear localization of Gwl contributes to its biochemical function, a number of possibilities are proposed. First, Gwl may phosphorylate its substrates in the nucleus to promote mitotic entry. Alternatively, Gwl may be initially activated in the nucleus prior to its nuclear export during the early stage of mitotic entry. The latter possibility may be attributed to the nuclear localization of its activating signal, such as MPF activity, or a lower level of inhibitory activity in the nucleus, such as phosphatases that target Gwl. Finally, Gwl negatively regulates the DNA damage checkpoint in Xenopus egg extracts. It is thus possible that the nuclear localization of Gwl contributes to mitotic entry by suppressing an inhibitory signal, such as the cell cycle checkpoint governing DNA damage or incomplete DNA replication (Yamamoto, 2014).

Cdc37 is essential for chromosome segregation and cytokinesis in higher eukaryotes

Cdc37 has been shown to be required for the activity and stability of protein kinases that regulate different stages of cell cycle progression. However, little is known so far regarding interactions of Cdc37 with kinases that play a role in cell division. Loss of function of Cdc37 in Drosophila leads to defects in mitosis and male meiosis, and these phenotypes closely resemble those brought about by the inactivation of Aurora B. Evidence is provided that Aurora B interacts with and requires the Cdc37/Hsp90 complex for its stability. It is concluded that the Cdc37/Hsp90 complex modulates the function of Aurora B and that most of the phenotypes brought about by the loss of Cdc37 function can be explained by the inactivation of this kinase. These observations substantiate the role of Cdc37 as an upstream regulatory element of key cell cycle kinases (Lange, 2002).

To investigate the abnormal phenotypes brought about by mutation in Cdc37, meiosis was followed in mutant spermatocytes by time-lapse video microscopy. At anaphase I mutant chromosomes are poorly condensed and often fail to align in a proper metaphase plate. The overall distance between the two centrosomes at metaphase is shorter than in control cells and the overall shape of the mutant spindle is stumpier. During metaphase, the homologs split apart asynchronously and the first signs of splitting are observed 5.9 min before the onset of poleward movement, earlier than in control cells (1.2 min). During anaphase, segregation mistakes are obvious. Some chromosomes acquire an amphitelic orientation, i.e., with both sister kinetochores orientated to opposite poles. Premature sister chromatid separation takes place as the amphitelic chromosomes segregate their chromatids during anaphase I. Single chromatids are also observed at different positions within the anaphase spindle. After segregation the chromatin decondenses and the daughter nuclei are formed. No sign of furrow constriction is detected and cytokinesis does not occur giving rise to binucleated cells that sometimes might contain additional micronuclei (Lange, 2002).

Having shown that Cdc37 is essential for chromosome segregation and cytokinesis in meiosis, the function of Cdc37 was investigated via an independent approach in mitotic cells. To this end, Cdc37 was ablated by RNAi in Drosophila SL2 cells and the results were compared with the phenotypes produced by Aurora B RNAi inactivation in these cells. Depletion of Cdc37 inhibits cell proliferation starting 3 days after transfection, Aurora B depletion inhibits proliferation already after 2 days, while control cells followed exponential growth. Phase contrast and immunofluorescence microscopy analysis revealed that most Cdc37 dsRNA-treated cells had grown abnormally large, i.e. 3-4 times the size of control cells and Aurora B dsRNA-treated cells increased more than 4 times in size. Both Cdc37 and Aurora B dsRNA treated cells contained multiple abnormally shaped and unequally sized nuclei indicating cytokinesis failure and problems in chromosome segregation. In addition, these cells also had an increased DNA content. Propidium iodine labelling and subsequent FACS analysis revealed that the majority of Cdc37 RNAi-treated cells (75%) had a 4N DNA content, while Aurora B RNAi-treated cells exhibit an even higher degree of ploidy. Thus, Cdc37 RNAi cells accomplish one round of DNA synthesis but undergo no further rounds of synthesis as detected in the Aurora B dsRNA-treated cells. These results are consistent with cytokinesis failure and problems in chromosome segregation seen in Cdc37 mutant spermatocytes. They are also consistent with the hypothesis that inactivation of Aurora B might be a major contributing factor to the phenotypes brought about by inactivation of Cdc37. Taken together, these results indicate that Cdc37 function is required for cell cycle progression and cytokinesis in meiotic and mitotic cells (Lange, 2002).

To determine whether Cdc37 and Aurora B are part of a molecular complex, co-immunoprecipitation assays were performed in mitotic extracts from mammalian tissue culture cells and in Drosophila embryonic extracts. Cdc37 was found to co-immunoprecipitate with Aurora B and Hsp90 in a number of different cell lines: NIH 3T3, a mouse fibroblast cell line; SW480, a colorectal adenocarcinoma cell line, and A549, a lung carcinoma cell line. This association is absent in cells that are treated with geldanamycin (GA), an inhibitor of the Hsp90/Cdc37 complex, indicating a functional relationship between Cdc37/Hsp90 and Aurora B. Interestingly, Aurora B binds less Cdc37 and Hsp90 in A549 cells when compared with the two other cell lines, and moreover, this interaction seems to be independent of GA treatment (Lange, 2002).

A general cytoplasmic and perinuclear localization of Cdc37 has been described previously. The cytoplasmic and perinuclear localization was confirmed by immunofluorescence microscopy in interphasic mammalian cells (NIH 3T3, SW480 and HeLa). However, using single labelling with anti-Cdc37 antibodies or double labelling together with an anti-α-tubulin antibody, a distinct labelling could be detected in the central spindle and in the midbody. Moreover, Cdc37 co-localizes with Aurora B on the spindle microtubules and midbody. In vitro microtubule-pelleting assays were performed to test whether this localization of Cdc37 could be due to microtubule binding in mitosis. Extracts from mitotically enriched HeLa and SW480 cells were incubated at 37°C in the presence of taxol to polymerize and stabilize microtubules from endogenous tubulin. Control extracts were incubated with nocodazole to depolymerize microtubules. The microtubules and associated proteins were subsequently pelleted. Only the microtubule-containing pellets carried Cdc37 while the pellets of the nocodazole-treated extracts did not, indicating a specific association of Cdc37 with microtubules. Some of the Cdc37 protein remained in the supernatant in the taxol-treated samples indicating that not all the pool of Cdc37 associates with microtubules. Microtubule pelleting was also carried out using interphase extracts, in which Cdc37 did not pellet with microtubules (Lange, 2002).

Altogether, the molecular and cytological data established that the function of Cdc37 and the Cdc37/Hsp90 complex are essential in wild-type cells to maintain stability of Aurora B in diverse tissues and cells of Drosophila and humans. Interfering with Cdc37 function leads to lack of a central spindle, aberrant chromosome segregation and cell cycle arrest. Interestingly, the interaction between Aurora B and Cdc37 is defective in certain cancer cells (Lange, 2002).

Mutations in Hsp83 and cdc37 impair signaling by the sevenless receptor tyrosine kinase in Drosophila

A highly conserved signal cascade functions subsequent to receptor tyrosine kinase activation. Signaling by the sevenless receptor, required for differentiation of the R7 photoreceptor neuron in Drosophila, is reduced by mutations in E(sev)3A and E(sev)3B. This study shows that E(sev)3A is a member of the Hsp90 family of stress proteins and that E(sev)3B encodes a homolog of the cell cycle control protein Cdc37 from S. cerevisiae. Mutations in E(sev)3B also dominantly enhance mutations in Dmcdc2, the gene encoding the p34 protein kinase that regulates the G2/M transition. Together, these data support a role for Hsp90 proteins in tyrosine kinase regulation and suggest that signals promoting neuronal differentiation may involve cell cycle control (Cutforth, 1994).


Functions of Cdc37 orthologs in other species

Cdc37 regulates Ryk signaling by stabilizing the cleaved Ryk intracellular domain

Ryk is a Wnt receptor that plays an important role in neurogenesis, neurite outgrowth, and axon guidance. It has been reported that the Ryk receptor is cleaved by gamma-secretase and that its intracellular domain (ICD) translocates to the nucleus upon Wnt stimulation. Cleavage of Ryk and its ICD is important for the function of Ryk in neurogenesis. However, the question of how the Ryk ICD is stabilized and translocated into the nucleus remains unanswered. This study shows that the Ryk ICD undergoes ubiquitination and proteasomal degradation. Cdc37, a subunit of the molecular chaperone Hsp90 complex, was identified as a Ryk ICD-interacting protein that inhibits proteasomal degradation of the Ryk ICD. Overexpression of Cdc37 increases Ryk ICD levels and promotes its nuclear localization, whereas Cdc37 knockdown reduces Ryk ICD stability. Furthermore, it was discovered that the Cdc37-Ryk ICD complex is disrupted during neural differentiation of embryonic stem cells, resulting in Ryk ICD degradation. These results suggest that Cdc37 plays an essential role in regulating Ryk ICD stability and therefore in Ryk-mediated signal transduction (Lyu, 2009).

Fused kinase is stabilized by Cdc37/Hsp90 and enhances Gli protein levels

Serine/threonine kinase Fused (Fu) is an essential component of Hedgehog (Hh) signaling in Drosophila, but the biochemical functions of Fu remain unclear. This study investigated proteins co-precipitated with mammalian Fu and identified a kinase-specific chaperone complex, Cdc37/Hsp90, as a novel-binding partner of Fu. Inhibition of Hsp90 function by geldanamycin (GA) induces rapid degradation of Fu through a ubiquitin-proteasome pathway. It was next shown that co-expression of Fu with transcription factors Gli1 and Gli2 significantly increases their protein levels and luciferase reporter activities, which are blocked by GA. These increases can be ascribed to Fu-mediated stabilization of Gli because co-expression of Fu prolongs half-life of Gli1 and reduces polyubiquitination of Gli1. Finally, it was shown that GA inhibits proliferation of PC3, a Hh signaling-activated prostate cancer cell line. This growth inhibition is partially rescued by expression of ectopic Gli1, suggesting that Fu may contribute to enhance Hh signaling activity in cancer cells (Kise, 2006).

Activity of Cdc2 and its interaction with the cyclin Cdc13 depend on the molecular chaperone Cdc37 in Schizosaccharomyces pombe

Cdc37 is a molecular chaperone whose clients are predominantly protein kinases, many of which are important in cell-cycle progression. Temperature-sensitive mutants of cdc37 in Schizosaccharomyces pombe are lethal at the restrictive temperature, arresting cell division within a single cell cycle. These mutant cells elongate during incubation at the restrictive temperature, consistent with a cell-cycle defect. The cell-cycle arrest arises from defective function of the mutant Cdc37 proteins rather than a reduction in Cdc37 protein levels. Around 80% of the arrested, elongated cells contain a single nucleus and replicated (2C) DNA content, indicating that these mutants arrest the cell cycle in G2 or mitosis (M). Cytological observations show that the majority of cells arrest in G2. In fission yeast, a G2 cell-cycle arrest can arise by inactivation of the cyclin-dependent kinase (Cdk) Cdc2 that regulates entry into mitosis. Studies of the cdc37 temperature-sensitive mutants show a genetic interaction with some cdc2 alleles and overexpression of cdc2 rescues the lethality of some cdc37 alleles at the restrictive temperature, suggesting that Cdc2 is a likely client for the Cdc37 molecular chaperone. In cdc37 temperature-sensitive mutants at the restrictive temperature, the level of Cdc2 protein remains constant but Cdc2 protein kinase activity is greatly reduced. Inactivation of Cdc2 appears to result from the inability to form complexes with its mitotic cyclin partner Cdc13. Further evidence for Cdc2 being a client of Cdc37 in S. pombe comes from the identification of genetic and biochemical interactions between these proteins (Turnbull, 2006).

p50(cdc37) binds directly to the catalytic domain of Raf as well as to a site on hsp90 that is topologically adjacent to the tetratricopeptide repeat binding site

Several protein kinases [e.g. pp60(src), v-Raf] exist in heterocomplexes with hsp90 and a 50-kDa protein that is the mammalian homolog of the yeast cell cycle control protein Cdc37. In contrast, unliganded steroid receptors exist in heterocomplexes with hsp90 and a tetratricopeptide repeat (TPR) domain protein, such as an immunophilin. Although p50(cdc37) and TPR domain proteins bind directly to hsp90, p50(cdc37) is not present in native steroid receptor.hsp90 heterocomplexes. To obtain some insight as to how v-Raf selects predominantly hsp90.p50(cdc37) heterocomplexes, rather than hsp90.TPR protein heterocomplexes, the binding of p50(cdc37) to hsp90 and to Raf was examined. p50(cdc37) exists in separate hsp90 heterocomplexes from the TPR domain proteins and intact TPR proteins compete for p50(cdc37) binding to hsp90, but a protein fragment containing a TPR domain does not compete. This suggests that the binding site for p50(cdc37) lies topologically adjacent to the TPR acceptor site on the surface of hsp90. Also, p50(cdc37) binds directly to v-Raf, with the catalytic domain of Raf being sufficient for binding. It is proposed that the combination of exclusive binding of p50(cdc37) versus a TPR domain protein to hsp90 plus direct binding of p50(cdc37) to Raf allows the protein kinase to select for the dominant hsp90.p50(cdc37) composition that is observed with a variety of protein kinase heterocomplexes immunoadsorbed from cytosols (Silverstein, 1998).

p50(cdc37) acting in concert with Hsp90 is required for Raf-1 function

Genetic screens in Drosophila have identified p50(cdc37) to be an essential component of the sevenless receptor/mitogen-activated kinase protein (MAPK) signaling pathway, but neither the function nor the target of p50(cdc37) in this pathway has been defined. Cdc37 is a protein kinase targeting subunit of heat shock protein 90. In this study, the role of p50(cdc37) and its Hsp90 chaperone partner in Raf/Mek/MAPK signaling was studied biochemically. Coexpression of wild-type p50(cdc37) with Raf-1 results in robust and dose-dependent activation of Raf-1 in Sf9 cells. In addition, p50(cdc37) greatly potentiates v-Src-mediated Raf-1 activation. p50(cdc37) is the primary determinant of Hsp90 recruitment to Raf-1. Overexpression of a p50(cdc37) mutant that is unable to recruit Hsp90 into the Raf-1 complex inhibits Raf-1 and MAPK activation by growth factors. Similarly, pretreatment with geldanamycin (GA), an Hsp90-specific inhibitor, prevents both the association of Raf-1 with the p50(cdc37)-Hsp90 heterodimer and Raf-1 kinase activation by serum. Activation of Raf-1 via baculovirus coexpression with oncogenic Src or Ras in Sf9 cells is also strongly inhibited by dominant negative p50(cdc37) or by GA. Thus, formation of a ternary Raf-1-p50(cdc37)-Hsp90 complex is crucial for Raf-1 activity and MAPK pathway signaling. These results provide the first biochemical evidence for the requirement of the p50(cdc37)-Hsp90 complex in protein kinase regulation and for Raf-1 function in particular (Grammatikakis. 1999).


REFERENCES

Search PubMed for articles about Drosophila Cdc37

Caplan, A. J., Ma'ayan, A. and Willis, I. M. (2007). Multiple kinases and system robustness: a link between Cdc37 and genome integrity. Cell Cycle 6(24): 3145-3147. PubMed ID: 18075309

Cutforth, T. and Rubin, G. M. (1994). Mutations in Hsp83 and cdc37 impair signaling by the sevenless receptor tyrosine kinase in Drosophila. Cell 77(7): 1027-1036. PubMed ID: 8020093

Grammatikakis. N., et al. (1999). p50(cdc37) acting in concert with Hsp90 is required for Raf-1 function. Mol. Cell. Biol. 19(3): 1661-72. PubMed ID: 10022854

Huang, J. and Wang, H. (2018). Hsp83/Hsp90 physically associates with Insulin receptor to promote neural stem cell reactivation. Stem Cell Reports 11(4): 883-896. PubMed ID: 30245208

Karnitz, L. M. and Felts, S. J. (2007). Cdc37 regulation of the kinome: when to hold 'em and when to fold 'em. Sci STKE 2007(385): pe22. PubMed ID: 17488976

Kise, Y., Takenaka, K., Tezuka, T., Yamamoto, T. and Miki, H. (2006). Fused kinase is stabilized by Cdc37/Hsp90 and enhances Gli protein levels. Biochem Biophys Res Commun 351(1): 78-84. PubMed ID: 17054904

Lange, B. M., Rebollo, E., Herold, A. and Gonzalez, C. (2002). Cdc37 is essential for chromosome segregation and cytokinesis in higher eukaryotes. EMBO J 21(20): 5364-5374. PubMed ID: 12374737

Lee, C. W., Kwon, Y. C., Lee, Y., Park, M. Y. and Choe, K. M. (2019). cdc37 is essential for JNK pathway activation and wound closure in Drosophila. Mol Biol Cell: mbcE18120822. PubMed ID: 31483695

Li, T., Jiang, H. L., Tong, Y. G. and Lu, J. J. (2018). Targeting the Hsp90-Cdc37-client protein interaction to disrupt Hsp90 chaperone machinery. J Hematol Oncol 11(1): 59. PubMed ID: 29699578

Lyu, J., Wesselschmidt, R. L. and Lu, W. (2009). Cdc37 regulates Ryk signaling by stabilizing the cleaved Ryk intracellular domain. J Biol Chem 284(19): 12940-12948. PubMed ID: 19269974

Martins, T., Maia, A. F., Steffensen, S. and Sunkel, C. E. (2009). Sgt1, a co-chaperone of Hsp90 stabilizes Polo and is required for centrosome organization. EMBO J 28(3): 234-247. PubMed ID: 19131964

Pearl, L. H. (2005). Hsp90 and Cdc37 -- a chaperone cancer conspiracy. Curr Opin Genet Dev 15(1): 55-61. PubMed ID: 15661534

Silverstein, A. M., et al. (1998). p50(cdc37) binds directly to the catalytic domain of Raf as well as to a site on hsp90 that is topologically adjacent to the tetratricopeptide repeat binding site. J. Biol. Chem. 273(32): 20090-5. PubMed ID: 9685350

Swarup, S., Pradhan-Sundd, T. and Verheyen, E. M. (2015). Genome-wide identification of phospho-regulators of Wnt signaling in Drosophila. Development 142(8): 1502-1515. PubMed ID: 25852200

Taipale, M., Jarosz, D. F. and Lindquist, S. (2010). HSP90 at the hub of protein homeostasis: emerging mechanistic insights. Nat Rev Mol Cell Biol 11(7): 515-528. PubMed ID: 20531426

Turnbull, E. L., Martin, I. V. and Fantes, P. A. (2006). Activity of Cdc2 and its interaction with the cyclin Cdc13 depend on the molecular chaperone Cdc37 in Schizosaccharomyces pombe. J Cell Sci 119(Pt 2): 292-302. PubMed ID: 16390871

Xiong, L., Zhao, T., Huang, X., Liu, Z. H., Zhao, H., Li, M. M., Wu, L. Y., Shu, H. B., Zhu, L. L. and Fan, M. (2009). Heat shock protein 90 is involved in regulation of hypoxia-driven proliferation of embryonic neural stem/progenitor cells. Cell Stress Chaperones 14(2): 183-192. PubMed ID: 18726712

Yamamoto, T. M., Wang, L., Fisher, L. A., Eckerdt, F. D. and Peng, A. (2014). Regulation of Greatwall kinase by protein stabilization and nuclear localization. Cell Cycle 13(22): 3565-3575. PubMed ID: 25483093


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date revised: 26 October 2019

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