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Wei-guo Zhu - One of the best experts on this subject based on the ideXlab platform.
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serine threonine kinase unc 51 like kinase 1 ulk1 phosphorylates the co chaperone cell division cycle protein 37 CDC37 and thereby disrupts the stability of CDC37 client proteins
Journal of Biological Chemistry, 2017Co-Authors: Fengjie Yuan, Luyao Zhang, Nan Zhang, Yanan Wang, Lina Wang, Wei-guo Zhu, Ying ZhaoAbstract:The serine/threonine kinase Unc-51-like kinase-1 (Ulk1) is thought to be essential for induction of autophagy, an intracellular bulk degradation process that is activated by various stresses. Although several proteins have been suggested as Ulk1 substrates during autophagic process, it still remains largely unknown about Ulk1's physiological substrates. Here, by performing in vitro and in vivo phosphorylation assay, we report that the co-chaperone cell division cycle protein 37 (CDC37) is a Ulk1 substrate. Ulk1-mediated phosphorylation of Ser-339 in CDC37 compromised the recruitment of client kinases to a complex comprising CDC37 and heat shock protein 90 (Hsp90) but only modestly affected CDC37 binding to Hsp90. Because the recruitment of protein kinase clients to the Hsp90 complex is essential for their stability and functions, Ser-339 phosphorylation of CDC37 disrupts its ability as a co-chaperone to coordinate Hsp90. Hsp90 inhibitors are cancer chemotherapeutic agents by inducing depletion of clients, many of which are oncogenes. Upon treatment with an Hsp90 inhibitor in cancer cells, Ulk1 promoted the degradation of Hsp90-CDC37 client kinases, resulting in increased cellular sensitivity to Hsp90 inhibitors. Thus, our study provides evidence for an anti-proliferative role of Ulk1 in response to Hsp90 inhibition in cancer cells.
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Serine/Threonine Kinase Unc-51-like Kinase-1 (Ulk1) Phosphorylates the Co-chaperone Cell Division Cycle Protein 37 (CDC37) and Thereby Disrupts the Stability of CDC37 Client Proteins.
The Journal of biological chemistry, 2017Co-Authors: Fengjie Yuan, Luyao Zhang, Nan Zhang, Yanan Wang, Lina Wang, Wei-guo ZhuAbstract:Abstract The serine/threonine kinase Unc-51-like kinase-1 (Ulk1) is thought to be essential for induction of autophagy, an intracellular bulk degradation process that is activated by various stresses. Although several proteins have been suggested as Ulk1 substrates during autophagic process, it still remains largely unknown about Ulk1's physiological substrates. Here, by performing in vitro and in vivo phosphorylation assay, we report that the co-chaperone cell division cycle protein 37 (CDC37) is a Ulk1 substrate. Ulk1-mediated phosphorylation of Ser-339 in CDC37 compromised the recruitment of client kinases to a complex comprising CDC37 and heat shock protein 90 (Hsp90) but only modestly affected CDC37 binding to Hsp90. Because the recruitment of protein kinase clients to the Hsp90 complex is essential for their stability and functions, Ser-339 phosphorylation of CDC37 disrupts its ability as a co-chaperone to coordinate Hsp90. Hsp90 inhibitors are cancer chemotherapeutic agents by inducing depletion of clients, many of which are oncogenes. Upon treatment with an Hsp90 inhibitor in cancer cells, Ulk1 promoted the degradation of Hsp90-CDC37 client kinases, resulting in increased cellular sensitivity to Hsp90 inhibitors. Thus, our study provides evidence for an anti-proliferative role of Ulk1 in response to Hsp90 inhibition in cancer cells.
Stuart K. Calderwood - One of the best experts on this subject based on the ideXlab platform.
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MZF1 and SCAND1 Reciprocally Regulate CDC37 Gene Expression in Prostate Cancer
Cancers, 2019Co-Authors: Takanori Eguchi, Thomas Prince, Manh Tien Tran, Chiharu Sogawa, Benjamin J. Lang, Stuart K. CalderwoodAbstract:Cell division control 37 (CDC37) increases the stability of heat shock protein 90 (HSP90) client proteins and is thus essential for numerous intracellular oncogenic signaling pathways, playing a key role in prostate oncogenesis. Notably, elevated expression of CDC37 was found in prostate cancer cells, although the regulatory mechanisms through which CDC37 expression becomes increased are unknown. Here we show both positive and negative regulation of CDC37 gene transcription by two members of the SREZBP-CTfin51-AW1-Number 18 cDNA (SCAN) transcription factor family-MZF1 and SCAND1, respectively. Consensus DNA-binding motifs for myeloid zinc finger 1 (MZF1/ZSCAN6) were abundant in the CDC37 promoter region. MZF1 became bound to these regulatory sites and trans-activated the CDC37 gene whereas MZF1 depletion decreased CDC37 transcription and reduced the tumorigenesis of prostate cancer cells. On the other hand, SCAND1, a zinc fingerless SCAN box protein that potentially inhibits MZF1, accumulated at MZF1-binding sites in the CDC37 gene, negatively regulated the CDC37 gene and inhibited tumorigenesis. SCAND1 was abundantly expressed in normal prostate cells but was reduced in prostate cancer cells, suggesting a potential tumor suppressor role of SCAND1 in prostate cancer. These findings indicate that CDC37, a crucial protein in prostate cancer progression, is regulated reciprocally by MZF1 and SCAND1.
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SCAND1 Suppresses CDC37 Gene Transcription by Repressing MZF1
2019Co-Authors: Takanori Eguchi, Thomas Prince, Chiharu Sogawa, Benjamin J. Lang, Tien Manh Tran, Stuart K. CalderwoodAbstract:Cell division control 37 (CDC37) increases the stability of HSP90 client proteins and is thus essential for numerous intracellular oncogenic signaling pathways, playing a key role in prostate oncogenesis. Notably, elevated expression of CDC37 was found in prostate cancer cells, although the regulatory mechanisms through which CDC37 expression becomes increased are unknown. Here we show both positive and negative regulation of CDC37 gene transcription by two members of the SCAN transcription factor family- MZF1 and SCAND1, respectively. Consensus DNA-binding motifs for myeloid zinc finger 1 (MZF1 / ZSCAN6) were abundant in the CDC37 promoter region. MZF1 became bound to these regulatory sites and trans-activated the CDC37 gene whereas MZF1 depletion decreased CDC37 transcription and reduced tumorigenesis of prostate cancer cells. On the other hand, SCAND1, a zinc-fingerless SCAN box protein that potentially inhibits MZF1, accumulated at MZF1-binding sites in CDC37 gene, negatively regulated CDC37 gene and inhibited tumorigenesis. SCAND1 was abundantly expressed in normal prostate cells but was reduced in prostate cancer cells, suggesting a potential tumor suppressor role of SCAND1 in prostate cancer. These findings indicate that CDC37, a crucial protein in prostate cancer progression, is regulated reciprocally by MZF1 and SCAND1.
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SCAND1 suppresses CDC37 gene transcription by repressing MZF1
2019Co-Authors: Takanori Eguchi, Thomas Prince, Manh Tien Tran, Chiharu Sogawa, Benjamin J. Lang, Stuart K. CalderwoodAbstract:Abstract CDC37 increases the stability of HSP90 client proteins and is essential for numerous intracellular oncogenic signaling pathways. Elevated expression of CDC37 was found in prostate cancer cells, although the regulatory mechanisms through which CDC37 expression becomes increased are unknown. Here we show both positive and negative regulation of CDC37 gene transcription by two members of the SCAN transcription factor family- MZF1 and SCAND1, respectively. Consensus DNA-binding motifs for MZF1 were abundant in the CDC37 promoter region. MZF1 became bound to these regulatory sites and trans-activated the CDC37 gene whereas MZF1 depletion decreased CDC37 transcrption and reduced tumorigenesis of prostate cancer cells. On the other hand, SCAND1, a zinc-fingerless SCAN box protein that potentially inhibits MZF1, accumulated at MZF1-binding sites in CDC37 gene, negatively regulated CDC37 gene and inhibited tumorigenesis. SCAND1 was abundantly expressed in normal prostate cells but was reduced in prostate cancer cells, suggesting a potential tumor suppressor role of SCAND1 in prostate cancer. These findings indicate that CDC37, a crucial protein in prostate cancer progression, is regulated reciprocally by MZF1 and SCAND1.
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CDC37 as a co-chaperone to Hsp90.
Sub-cellular biochemistry, 2014Co-Authors: Stuart K. CalderwoodAbstract:The co-chaperone p50/CDC37 is an important partner for Hsp90, assisting in molecular chaperone activities, particularly with regard to the regulation of protein kinases. The Hsp90/CDC37complex controls the folding of a large proportion of protein kinases and thus stands at the hub of a multitude of intracellular signaling networks. Its effects thus reach beyond the housekeeping pathways of protein folding into regulation of a wide range of cellular processes. Due to its influence in cell growth pathways CDC37 has attracted much attention as a potential intermediate in carcinogenesis. CDC37 is an attractive potential target in cancer due to: (1) it may be expressed to high level in some types of cancer and (2) CDC37 controls multiple signaling pathways. This indicates a potential for: (1) selectivity due to its elevated expression and (2) robustness as the co-chaperone may control multiple growth signaling pathways and thus be less prone to evolution of resistance than other oncoproteins. CDC37 may also be involved in other aspects of pathophysiology. Protein aggregation disorders have been linked to molecular chaperones and to age related declines in molecular chaperones and co-chaperones. CDC37 appears to be a potential agent in longevity due to its links to protein folding and autophagy and it will be informative to study the role of CDC37 maintenance/decline in aging organisms.
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Targeting the oncogene and kinome chaperone CDC37
Nature reviews. Cancer, 2008Co-Authors: Phillip J. Gray, Thomas Prince, Mary Ann Stevenson, Jinrong Cheng, Stuart K. CalderwoodAbstract:CDC37 is a molecular chaperone that physically stabilizes the catalytic domains found in protein kinases and is therefore a wide-spectrum regulator of protein phosphorylation. It is also an overexpressed oncoprotein that mediates carcinogenesis by stabilizing the compromised structures of mutant and/or overexpressed oncogenic kinases. Recent work shows that such dependency of malignant cells on increased CDC37 expression is a vulnerability that can be targeted in cancer by agents that deplete or inhibit CDC37. CDC37 is thus a candidate for broad-spectrum molecular cancer therapy.
Eisuke Nishida - One of the best experts on this subject based on the ideXlab platform.
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Evaluating CK2 activity with the antibody specific for the CK2-phosphorylated form of a kinase-targeting cochaperone CDC37.
Molecular and cellular biochemistry, 2008Co-Authors: Yoshihiko Miyata, Eisuke NishidaAbstract:CK2-dependent phosphorylation of a kinase-specific Hsp90 co-chaperone CDC37 on a conserved serine residue (Ser13) is essential for the function of CDC37 [Bandhakavi S. et al. J. Biol. Chem. 278:2829-2836, 2003; Shao J. et al. J. Biol. Chem. 278:38117-38220, 2003; Miyata Y., & Nishida E. Mol. Cell. Biol. 24:4065-4074, 2004]. We have recently produced an anti-[pSer13]-CDC37 antibody which specifically recognizes CDC37 that is phosphorylated on Ser 13 [Miyata Y. & Nishida E. FEBS J. 274:5690-5703, 2007]. Here we investigated CK2 activity both in vitro and in cultured cells by using anti-[pSer13]-CDC37 antibody. Immunoblotting with this antibody showed that heparin and 4,5,6,7-tetrabromobenzotriazole (TBB), known CK2 inhibitors, inhibited in vitro phosphorylation of CDC37 on Ser13 by CK2 holoenzyme or CK2alpha, confirming the specificity of the antibody to detect CK2 activity. Treatment of cells with TBB resulted in the decrease in the phosphorylation level of endogenous CDC37 on Ser13, as revealed by anti-[pSer13]-CDC37, and overexpression of either CK2alpha or CK2beta subunit enhanced the CDC37 phosphorylation level. While CK2 is suggested to be involved in cell proliferation, mitogenic stimulation of starved cells by fresh serum or insulin-like growth factor-I did not enhance phosphorylation of CDC37 on Ser13. CK2 inhibitors are known to induce cell apoptosis, suggesting a reverse correlation between cell apoptosis and CK2 activity. However, cellular apoptotic stresses, such as anisomycin treatment and UV irradiation, were found to rather modestly increase phosphorylation of CDC37 on Ser13. These results show that the anti-[pSer13]-CDC37 antibody can be a promising new tool to evaluate in vivo CK2 activity.
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Evaluating CK2 activity with the antibody specific for the CK2-phosphorylated form of a kinase-targeting cochaperone CDC37.
Molecular and Cellular Biochemistry, 2008Co-Authors: Yoshihiko Miyata, Eisuke NishidaAbstract:CK2-dependent phosphorylation of a kinase-specific Hsp90 co-chaperone CDC37 on a conserved serine residue (Ser13) is essential for the function of CDC37 [Bandhakavi S. et al. J. Biol. Chem. 278:2829–2836, 2003; Shao J. et al. J. Biol. Chem. 278:38117–38220, 2003; Miyata Y., & Nishida E. Mol. Cell. Biol. 24:4065–4074, 2004]. We have recently produced an anti-[pSer13]-CDC37 antibody which specifically recognizes CDC37 that is phosphorylated on Ser 13 [Miyata Y. & Nishida E. FEBS J. 274:5690–5703, 2007]. Here we investigated CK2 activity both in vitro and in cultured cells by using anti-[pSer13]-CDC37 antibody. Immunoblotting with this antibody showed that heparin and 4,5,6,7-tetrabromobenzotriazole (TBB), known CK2 inhibitors, inhibited in vitro phosphorylation of CDC37 on Ser13 by CK2 holoenzyme or CK2α, confirming the specificity of the antibody to detect CK2 activity. Treatment of cells with TBB resulted in the decrease in the phosphorylation level of endogenous CDC37 on Ser13, as revealed by anti-[pSer13]-CDC37, and overexpression of either CK2α or CK2β subunit enhanced the CDC37 phosphorylation level. While CK2 is suggested to be involved in cell proliferation, mitogenic stimulation of starved cells by fresh serum or insulin-like growth factor-I did not enhance phosphorylation of CDC37 on Ser13. CK2 inhibitors are known to induce cell apoptosis, suggesting a reverse correlation between cell apoptosis and CK2 activity. However, cellular apoptotic stresses, such as anisomycin treatment and UV irradiation, were found to rather modestly increase phosphorylation of CDC37 on Ser13. These results show that the anti-[pSer13]-CDC37 antibody can be a promising new tool to evaluate in vivo CK2 activity.
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Analysis of the CK2-dependent phosphorylation of serine 13 in CDC37 using a phospho-specific antibody and phospho-affinity gel electrophoresis.
The FEBS journal, 2007Co-Authors: Yoshihiko Miyata, Eisuke NishidaAbstract:The CK2-dependent phosphorylation of Ser13 in cell division cycle protein 37 (CDC37), a kinase-specific heat shock protein 90 (Hsp90) cochaperone, has previously been reported to be essential for the association of CDC37 with signaling protein kinases [Bandhakavi S, McCann RO, Hanna DE & Glover CVC (2003) J Biol Chem278, 2829–2836; Shao J, Prince T, Hartson SD & Matts RL (2003) J Biol Chem278, 38117–38220; Miyata Y & Nishida E (2004) Mol Cell Biol24, 4065–4074]. Here we describe a new phospho-specific antibody against CDC37 that recognizes recombinant purified CDC37 only when incubated with CK2 in the presence of Mg2+ and ATP. The replacement of Ser13 in CDC37 by nonphosphorylatable amino acids abolished binding to this antibody. The antibody was specific for phosphorylated CDC37 and did not crossreact with other CK2 substrates such as Hsp90 and FK506-binding protein 52. Using this antibody, we showed that complexes of Hsp90 with its client signaling kinases, Cdk4, MOK, v-Src, and Raf1, contained the CK2-phosphorylated form of CDC37 in vivo. Immunofluorescent staining showed that Hsp90 and the phosphorylated form of CDC37 accumulated in epidermal growth factor-induced membrane ruffles. We further characterized the phosphorylation of CDC37 using phospho-affinity gel electrophoresis. Our analyses demonstrated that the CK2-dependent phosphorylation of CDC37 on Ser13 caused a specific gel mobility shift, and that CDC37 in the complexes between Hsp90 and its client signaling protein kinases was in the phosphorylated form. Our results show the physiological importance of CK2-dependent CDC37 phosphorylation and the usefulness of phospho-affinity gel electrophoresis in protein phosphorylation analysis.
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CK2 binds, phosphorylates, and regulates its pivotal substrate CDC37, an Hsp90-cochaperone.
Molecular and Cellular Biochemistry, 2005Co-Authors: Yoshihiko Miyata, Eisuke NishidaAbstract:Protein kinase CK2 phosphorylates and regulates a large number of substrates but roles of CK2 in protein kinase-mediated signal transduction systems remain largely uncertain. CDC37 is a protein kinase-targeting molecular chaperone and its function in cooperation with Hsp90 is required for various signaling kinases. In this article, interaction between CK2 and CDC37 is described. We present evidence indicating that phosphorylation of CDC37 by CK2 in conserved Ser13 in the N-terminal extremity was prerequisite for the efficient binding activity of CDC37 to protein kinases including Akt, Cdk4, MOK, and Raf1. In addition, the phosphorylation of CDC37 by CK2 was crucial for the recruitment of Hsp90 to the protein kinase-CDC37 complexes. We observed that a subset of CK2 was associated with Hsp90 and CDC37 in cells. Whereas Hsp90 and CDC37 were exclusively distributed in the cytoplasm, CK2α and CK2β were localized mainly in the nucleus but also in the cytoplasm with different patterns. Moreover, direct association of CDC37 with CK2α was observed in an E. coli system. Collectively, these findings indicated that a subpopulation of CK2 forms complexes with Hsp90 and CDC37 in the cytoplasm and phosphorylates CDC37, thus regulates the molecular chaperone activity of CDC37. Since CK2 activity depends on CDC37, CK2 and CDC37 constitute a positive feedback machinery to control multiple CDC37-dependent signaling protein kinases. The structure of CDC37 and physiological importance of the CK2-CDC37 interaction are discussed.
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CK2 binds, phosphorylates, and regulates its pivotal substrate CDC37, an Hsp90-cochaperone.
Molecular and cellular biochemistry, 2005Co-Authors: Yoshihiko Miyata, Eisuke NishidaAbstract:Protein kinase CK2 phosphorylates and regulates a large number of substrates but roles of CK2 in protein kinase-mediated signal transduction systems remain largely uncertain. CDC37 is a protein kinase-targeting molecular chaperone and its function in cooperation with Hsp90 is required for various signaling kinases. In this article, interaction between CK2 and CDC37 is described. We present evidence indicating that phosphorylation of CDC37 by CK2 in conserved Ser13 in the N-terminal extremity was prerequisite for the efficient binding activity of CDC37 to protein kinases including Akt, Cdk4, MOK, and Raf1. In addition, the phosphorylation of CDC37 by CK2 was crucial for the recruitment of Hsp90 to the protein kinase-CDC37 complexes. We observed that a subset of CK2 was associated with Hsp90 and CDC37 in cells. Whereas Hsp90 and CDC37 were exclusively distributed in the cytoplasm, CK2alpha and CK2beta were localized mainly in the nucleus but also in the cytoplasm with different patterns. Moreover, direct association of CDC37 with CK2alpha was observed in an E. coli system. Collectively, these findings indicated that a subpopulation of CK2 forms complexes with Hsp90 and CDC37 in the cytoplasm and phosphorylates CDC37, thus regulates the molecular chaperone activity of CDC37. Since CK2 activity depends on CDC37, CK2 and CDC37 constitute a positive feedback machinery to control multiple CDC37-dependent signaling protein kinases. The structure of CDC37 and physiological importance of the CK2-CDC37 interaction are discussed.
Yoshihiko Miyata - One of the best experts on this subject based on the ideXlab platform.
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Structural characterization of the N-terminal kinase-interacting domain of an Hsp90-cochaperone CDC37 by CD and solution NMR spectroscopy
Biochimica et biophysica acta. Proteins and proteomics, 2019Co-Authors: Futoshi Ihama, Yoshihiko Miyata, Mami Yamamoto, Chojiro Kojima, Toshimichi Fujiwara, Katsumi Matsuzaki, Masaru HoshinoAbstract:Abstract CDC37 is a protein kinase-targeting molecular chaperone, which cooperates with Hsp90 to assist the folding, assembly and maturation of various signaling kinases. It consists of three distinct domains: the N-terminal, middle, and C-terminal domain. While the middle domain is an Hsp90-binding domain, the N-terminal domain is recognized as a kinase-interacting domain. The N-terminal domain contains a well-conserved Ser residue at position 13, and the phosphorylation at this site has been shown to be a prerequisite for the interaction between CDC37 and signaling kinases. Although the phosphorylation of Ser13 might induce some conformational change in CDC37 molecule, little is known about the structure of the N-terminal domain of CDC37. We examined the structural and dynamic properties of several fragment proteins corresponding to the N-terminal region of CDC37 by circular dichroism and solution NMR spectroscopy. We found that the N-terminal domain of CDC37 exhibits highly dynamic structure, and it exists in the equilibrium between α-helical and more disordered structures. We also found that phosphorylation at Ser13 did not significantly change the overall structure of N-terminal fragment protein of CDC37. The results suggested that more complicated mechanisms might be necessary to explain the phosphorylation-activated interaction of CDC37 with various kinases.
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Evaluating CK2 activity with the antibody specific for the CK2-phosphorylated form of a kinase-targeting cochaperone CDC37.
Molecular and cellular biochemistry, 2008Co-Authors: Yoshihiko Miyata, Eisuke NishidaAbstract:CK2-dependent phosphorylation of a kinase-specific Hsp90 co-chaperone CDC37 on a conserved serine residue (Ser13) is essential for the function of CDC37 [Bandhakavi S. et al. J. Biol. Chem. 278:2829-2836, 2003; Shao J. et al. J. Biol. Chem. 278:38117-38220, 2003; Miyata Y., & Nishida E. Mol. Cell. Biol. 24:4065-4074, 2004]. We have recently produced an anti-[pSer13]-CDC37 antibody which specifically recognizes CDC37 that is phosphorylated on Ser 13 [Miyata Y. & Nishida E. FEBS J. 274:5690-5703, 2007]. Here we investigated CK2 activity both in vitro and in cultured cells by using anti-[pSer13]-CDC37 antibody. Immunoblotting with this antibody showed that heparin and 4,5,6,7-tetrabromobenzotriazole (TBB), known CK2 inhibitors, inhibited in vitro phosphorylation of CDC37 on Ser13 by CK2 holoenzyme or CK2alpha, confirming the specificity of the antibody to detect CK2 activity. Treatment of cells with TBB resulted in the decrease in the phosphorylation level of endogenous CDC37 on Ser13, as revealed by anti-[pSer13]-CDC37, and overexpression of either CK2alpha or CK2beta subunit enhanced the CDC37 phosphorylation level. While CK2 is suggested to be involved in cell proliferation, mitogenic stimulation of starved cells by fresh serum or insulin-like growth factor-I did not enhance phosphorylation of CDC37 on Ser13. CK2 inhibitors are known to induce cell apoptosis, suggesting a reverse correlation between cell apoptosis and CK2 activity. However, cellular apoptotic stresses, such as anisomycin treatment and UV irradiation, were found to rather modestly increase phosphorylation of CDC37 on Ser13. These results show that the anti-[pSer13]-CDC37 antibody can be a promising new tool to evaluate in vivo CK2 activity.
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Evaluating CK2 activity with the antibody specific for the CK2-phosphorylated form of a kinase-targeting cochaperone CDC37.
Molecular and Cellular Biochemistry, 2008Co-Authors: Yoshihiko Miyata, Eisuke NishidaAbstract:CK2-dependent phosphorylation of a kinase-specific Hsp90 co-chaperone CDC37 on a conserved serine residue (Ser13) is essential for the function of CDC37 [Bandhakavi S. et al. J. Biol. Chem. 278:2829–2836, 2003; Shao J. et al. J. Biol. Chem. 278:38117–38220, 2003; Miyata Y., & Nishida E. Mol. Cell. Biol. 24:4065–4074, 2004]. We have recently produced an anti-[pSer13]-CDC37 antibody which specifically recognizes CDC37 that is phosphorylated on Ser 13 [Miyata Y. & Nishida E. FEBS J. 274:5690–5703, 2007]. Here we investigated CK2 activity both in vitro and in cultured cells by using anti-[pSer13]-CDC37 antibody. Immunoblotting with this antibody showed that heparin and 4,5,6,7-tetrabromobenzotriazole (TBB), known CK2 inhibitors, inhibited in vitro phosphorylation of CDC37 on Ser13 by CK2 holoenzyme or CK2α, confirming the specificity of the antibody to detect CK2 activity. Treatment of cells with TBB resulted in the decrease in the phosphorylation level of endogenous CDC37 on Ser13, as revealed by anti-[pSer13]-CDC37, and overexpression of either CK2α or CK2β subunit enhanced the CDC37 phosphorylation level. While CK2 is suggested to be involved in cell proliferation, mitogenic stimulation of starved cells by fresh serum or insulin-like growth factor-I did not enhance phosphorylation of CDC37 on Ser13. CK2 inhibitors are known to induce cell apoptosis, suggesting a reverse correlation between cell apoptosis and CK2 activity. However, cellular apoptotic stresses, such as anisomycin treatment and UV irradiation, were found to rather modestly increase phosphorylation of CDC37 on Ser13. These results show that the anti-[pSer13]-CDC37 antibody can be a promising new tool to evaluate in vivo CK2 activity.
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Analysis of the CK2-dependent phosphorylation of serine 13 in CDC37 using a phospho-specific antibody and phospho-affinity gel electrophoresis.
The FEBS journal, 2007Co-Authors: Yoshihiko Miyata, Eisuke NishidaAbstract:The CK2-dependent phosphorylation of Ser13 in cell division cycle protein 37 (CDC37), a kinase-specific heat shock protein 90 (Hsp90) cochaperone, has previously been reported to be essential for the association of CDC37 with signaling protein kinases [Bandhakavi S, McCann RO, Hanna DE & Glover CVC (2003) J Biol Chem278, 2829–2836; Shao J, Prince T, Hartson SD & Matts RL (2003) J Biol Chem278, 38117–38220; Miyata Y & Nishida E (2004) Mol Cell Biol24, 4065–4074]. Here we describe a new phospho-specific antibody against CDC37 that recognizes recombinant purified CDC37 only when incubated with CK2 in the presence of Mg2+ and ATP. The replacement of Ser13 in CDC37 by nonphosphorylatable amino acids abolished binding to this antibody. The antibody was specific for phosphorylated CDC37 and did not crossreact with other CK2 substrates such as Hsp90 and FK506-binding protein 52. Using this antibody, we showed that complexes of Hsp90 with its client signaling kinases, Cdk4, MOK, v-Src, and Raf1, contained the CK2-phosphorylated form of CDC37 in vivo. Immunofluorescent staining showed that Hsp90 and the phosphorylated form of CDC37 accumulated in epidermal growth factor-induced membrane ruffles. We further characterized the phosphorylation of CDC37 using phospho-affinity gel electrophoresis. Our analyses demonstrated that the CK2-dependent phosphorylation of CDC37 on Ser13 caused a specific gel mobility shift, and that CDC37 in the complexes between Hsp90 and its client signaling protein kinases was in the phosphorylated form. Our results show the physiological importance of CK2-dependent CDC37 phosphorylation and the usefulness of phospho-affinity gel electrophoresis in protein phosphorylation analysis.
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CK2 binds, phosphorylates, and regulates its pivotal substrate CDC37, an Hsp90-cochaperone.
Molecular and Cellular Biochemistry, 2005Co-Authors: Yoshihiko Miyata, Eisuke NishidaAbstract:Protein kinase CK2 phosphorylates and regulates a large number of substrates but roles of CK2 in protein kinase-mediated signal transduction systems remain largely uncertain. CDC37 is a protein kinase-targeting molecular chaperone and its function in cooperation with Hsp90 is required for various signaling kinases. In this article, interaction between CK2 and CDC37 is described. We present evidence indicating that phosphorylation of CDC37 by CK2 in conserved Ser13 in the N-terminal extremity was prerequisite for the efficient binding activity of CDC37 to protein kinases including Akt, Cdk4, MOK, and Raf1. In addition, the phosphorylation of CDC37 by CK2 was crucial for the recruitment of Hsp90 to the protein kinase-CDC37 complexes. We observed that a subset of CK2 was associated with Hsp90 and CDC37 in cells. Whereas Hsp90 and CDC37 were exclusively distributed in the cytoplasm, CK2α and CK2β were localized mainly in the nucleus but also in the cytoplasm with different patterns. Moreover, direct association of CDC37 with CK2α was observed in an E. coli system. Collectively, these findings indicated that a subpopulation of CK2 forms complexes with Hsp90 and CDC37 in the cytoplasm and phosphorylates CDC37, thus regulates the molecular chaperone activity of CDC37. Since CK2 activity depends on CDC37, CK2 and CDC37 constitute a positive feedback machinery to control multiple CDC37-dependent signaling protein kinases. The structure of CDC37 and physiological importance of the CK2-CDC37 interaction are discussed.
Fengjie Yuan - One of the best experts on this subject based on the ideXlab platform.
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serine threonine kinase unc 51 like kinase 1 ulk1 phosphorylates the co chaperone cell division cycle protein 37 CDC37 and thereby disrupts the stability of CDC37 client proteins
Journal of Biological Chemistry, 2017Co-Authors: Fengjie Yuan, Luyao Zhang, Nan Zhang, Yanan Wang, Lina Wang, Wei-guo Zhu, Ying ZhaoAbstract:The serine/threonine kinase Unc-51-like kinase-1 (Ulk1) is thought to be essential for induction of autophagy, an intracellular bulk degradation process that is activated by various stresses. Although several proteins have been suggested as Ulk1 substrates during autophagic process, it still remains largely unknown about Ulk1's physiological substrates. Here, by performing in vitro and in vivo phosphorylation assay, we report that the co-chaperone cell division cycle protein 37 (CDC37) is a Ulk1 substrate. Ulk1-mediated phosphorylation of Ser-339 in CDC37 compromised the recruitment of client kinases to a complex comprising CDC37 and heat shock protein 90 (Hsp90) but only modestly affected CDC37 binding to Hsp90. Because the recruitment of protein kinase clients to the Hsp90 complex is essential for their stability and functions, Ser-339 phosphorylation of CDC37 disrupts its ability as a co-chaperone to coordinate Hsp90. Hsp90 inhibitors are cancer chemotherapeutic agents by inducing depletion of clients, many of which are oncogenes. Upon treatment with an Hsp90 inhibitor in cancer cells, Ulk1 promoted the degradation of Hsp90-CDC37 client kinases, resulting in increased cellular sensitivity to Hsp90 inhibitors. Thus, our study provides evidence for an anti-proliferative role of Ulk1 in response to Hsp90 inhibition in cancer cells.
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Serine/Threonine Kinase Unc-51-like Kinase-1 (Ulk1) Phosphorylates the Co-chaperone Cell Division Cycle Protein 37 (CDC37) and Thereby Disrupts the Stability of CDC37 Client Proteins.
The Journal of biological chemistry, 2017Co-Authors: Fengjie Yuan, Luyao Zhang, Nan Zhang, Yanan Wang, Lina Wang, Wei-guo ZhuAbstract:Abstract The serine/threonine kinase Unc-51-like kinase-1 (Ulk1) is thought to be essential for induction of autophagy, an intracellular bulk degradation process that is activated by various stresses. Although several proteins have been suggested as Ulk1 substrates during autophagic process, it still remains largely unknown about Ulk1's physiological substrates. Here, by performing in vitro and in vivo phosphorylation assay, we report that the co-chaperone cell division cycle protein 37 (CDC37) is a Ulk1 substrate. Ulk1-mediated phosphorylation of Ser-339 in CDC37 compromised the recruitment of client kinases to a complex comprising CDC37 and heat shock protein 90 (Hsp90) but only modestly affected CDC37 binding to Hsp90. Because the recruitment of protein kinase clients to the Hsp90 complex is essential for their stability and functions, Ser-339 phosphorylation of CDC37 disrupts its ability as a co-chaperone to coordinate Hsp90. Hsp90 inhibitors are cancer chemotherapeutic agents by inducing depletion of clients, many of which are oncogenes. Upon treatment with an Hsp90 inhibitor in cancer cells, Ulk1 promoted the degradation of Hsp90-CDC37 client kinases, resulting in increased cellular sensitivity to Hsp90 inhibitors. Thus, our study provides evidence for an anti-proliferative role of Ulk1 in response to Hsp90 inhibition in cancer cells.