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Involvement of maternal embryonic leucine zipper kinase (MELK) in mammary carcinogenesis through interaction with Bcl-G, a pro-apoptotic member of the Bcl-2 family.

Lin ML, Park JH, Nishidate T, Nakamura Y, Katagiri T - Breast Cancer Res. (2007)

Bottom Line: Northern blot analyses on multiple human tissues and cancer cell lines demonstrated that MELK was overexpressed at a significantly high level in a great majority of breast cancers and cell lines, but was not expressed in normal vital organs (heart, liver, lung and kidney).We also found that MELK physically interacted with Bcl-GL through its amino-terminal region.Our findings suggest that the kinase activity of MELK is likely to affect mammary carcinogenesis through inhibition of the pro-apoptotic function of Bcl-GL.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Molecular Medicine, Human Genome Center, Institute of Medical Science, The University of Tokyo, Tokyo, Japan.

ABSTRACT

Introduction: Cancer therapies directed at specific molecular targets in signaling pathways of cancer cells, such as tamoxifen, aromatase inhibitors and trastuzumab, have proven useful for treatment of advanced breast cancers. However, increased risk of endometrial cancer with long-term tamoxifen administration and of bone fracture due to osteoporosis in postmenopausal women undergoing aromatase inhibitor therapy are recognized side effects. These side effects as well as drug resistance make it necessary to search for novel molecular targets for drugs on the basis of well-characterized mechanisms of action.

Methods: Using accurate genome-wide expression profiles of breast cancers, we found maternal embryonic leucine-zipper kinase (MELK) to be significantly overexpressed in the great majority of breast cancer cells. To assess whether MELK has a role in mammary carcinogenesis, we knocked down the expression of endogenous MELK in breast cancer cell lines using mammalian vector-based RNA interference. Furthermore, we identified a long isoform of Bcl-G (Bcl-GL), a pro-apoptotic member of the Bcl-2 family, as a possible substrate for MELK by pull-down assay with recombinant wild-type and kinase-dead MELK. Finally, we performed TUNEL assays and FACS analysis, measuring proportions of apoptotic cells, to investigate whether MELK is involved in the apoptosis cascade through the Bcl-GL-related pathway.

Results: Northern blot analyses on multiple human tissues and cancer cell lines demonstrated that MELK was overexpressed at a significantly high level in a great majority of breast cancers and cell lines, but was not expressed in normal vital organs (heart, liver, lung and kidney). Suppression of MELK expression by small interfering RNA significantly inhibited growth of human breast cancer cells. We also found that MELK physically interacted with Bcl-GL through its amino-terminal region. Immunocomplex kinase assay showed that Bcl-GL was specifically phosphorylated by MELK in vitro. TUNEL assays and FACS analysis revealed that overexpression of wild-type MELK suppressed Bcl-GL-induced apoptosis, while that of D150A-MELK did not.

Conclusion: Our findings suggest that the kinase activity of MELK is likely to affect mammary carcinogenesis through inhibition of the pro-apoptotic function of Bcl-GL. The kinase activity of MELK could be a promising molecular target for development of therapy for patients with breast cancers.

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MELK phosphorylates Bcl-GL in vitro. (a) Immunoprecipitates were subjected to immune complex kinase assay with wild-type (WT)-MELK or kinase-dead (D150A)-MELK). The single arrowhead indicates phosphorylated Bcl-GL, and the double arrowhead points to an autophosphorylated MELK protein. (b) Phosphorylation of a bacterial glutathione S-transferase (GST) Bcl-GL fusion recombinant protein (GST-Bcl-GL) by His-tagged WT-MELK (WT). The single arrowhead indicates phosphorylated GST-Bcl-GL protein, and the double arrowhead indicates an autophosphorylated His-tagged MELK protein. (c) In vitro phosphorylation of various partial amino-terminal constructs of Bcl-GL (N-1, N-2, N-3 and N-4; see Figure 3e) and full-length Bcl-GL (FL) by MELK. The single arrowheads indicate phosphorylated immunoprecipitated Bcl-GL proteins, and the double arrowhead indicates an autophosphorylated MELK recombinant protein.
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Figure 4: MELK phosphorylates Bcl-GL in vitro. (a) Immunoprecipitates were subjected to immune complex kinase assay with wild-type (WT)-MELK or kinase-dead (D150A)-MELK). The single arrowhead indicates phosphorylated Bcl-GL, and the double arrowhead points to an autophosphorylated MELK protein. (b) Phosphorylation of a bacterial glutathione S-transferase (GST) Bcl-GL fusion recombinant protein (GST-Bcl-GL) by His-tagged WT-MELK (WT). The single arrowhead indicates phosphorylated GST-Bcl-GL protein, and the double arrowhead indicates an autophosphorylated His-tagged MELK protein. (c) In vitro phosphorylation of various partial amino-terminal constructs of Bcl-GL (N-1, N-2, N-3 and N-4; see Figure 3e) and full-length Bcl-GL (FL) by MELK. The single arrowheads indicate phosphorylated immunoprecipitated Bcl-GL proteins, and the double arrowhead indicates an autophosphorylated MELK recombinant protein.

Mentions: To examine whether the Bcl-GL protein is a substrate of MELK kinase activity, we performed an immune complex kinase assay. We first confirmed the exogenous expression of Flag-tagged Bcl-GL (Flag-Bcl-GL) by western blot analysis (Figure 3f). As shown in Figure 4a, WT-MELK could phosphorylate Bcl-GL, but D150A-MELK could not (single arrowhead). In addition, autophosphorylation of an approximately 75 kDa protein of MELK was observed (Figure 4a; double arrowheads). Furthermore, we also confirmed that WT-MELK could phosphorylate GST-Bcl-GL but D150A-MELK could not (Figure 4b), indicating MELK can directly phosphorylate Bcl-GL. These findings suggest that Bcl-GL is a potential substrate for MELK.


Involvement of maternal embryonic leucine zipper kinase (MELK) in mammary carcinogenesis through interaction with Bcl-G, a pro-apoptotic member of the Bcl-2 family.

Lin ML, Park JH, Nishidate T, Nakamura Y, Katagiri T - Breast Cancer Res. (2007)

MELK phosphorylates Bcl-GL in vitro. (a) Immunoprecipitates were subjected to immune complex kinase assay with wild-type (WT)-MELK or kinase-dead (D150A)-MELK). The single arrowhead indicates phosphorylated Bcl-GL, and the double arrowhead points to an autophosphorylated MELK protein. (b) Phosphorylation of a bacterial glutathione S-transferase (GST) Bcl-GL fusion recombinant protein (GST-Bcl-GL) by His-tagged WT-MELK (WT). The single arrowhead indicates phosphorylated GST-Bcl-GL protein, and the double arrowhead indicates an autophosphorylated His-tagged MELK protein. (c) In vitro phosphorylation of various partial amino-terminal constructs of Bcl-GL (N-1, N-2, N-3 and N-4; see Figure 3e) and full-length Bcl-GL (FL) by MELK. The single arrowheads indicate phosphorylated immunoprecipitated Bcl-GL proteins, and the double arrowhead indicates an autophosphorylated MELK recombinant protein.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1851384&req=5

Figure 4: MELK phosphorylates Bcl-GL in vitro. (a) Immunoprecipitates were subjected to immune complex kinase assay with wild-type (WT)-MELK or kinase-dead (D150A)-MELK). The single arrowhead indicates phosphorylated Bcl-GL, and the double arrowhead points to an autophosphorylated MELK protein. (b) Phosphorylation of a bacterial glutathione S-transferase (GST) Bcl-GL fusion recombinant protein (GST-Bcl-GL) by His-tagged WT-MELK (WT). The single arrowhead indicates phosphorylated GST-Bcl-GL protein, and the double arrowhead indicates an autophosphorylated His-tagged MELK protein. (c) In vitro phosphorylation of various partial amino-terminal constructs of Bcl-GL (N-1, N-2, N-3 and N-4; see Figure 3e) and full-length Bcl-GL (FL) by MELK. The single arrowheads indicate phosphorylated immunoprecipitated Bcl-GL proteins, and the double arrowhead indicates an autophosphorylated MELK recombinant protein.
Mentions: To examine whether the Bcl-GL protein is a substrate of MELK kinase activity, we performed an immune complex kinase assay. We first confirmed the exogenous expression of Flag-tagged Bcl-GL (Flag-Bcl-GL) by western blot analysis (Figure 3f). As shown in Figure 4a, WT-MELK could phosphorylate Bcl-GL, but D150A-MELK could not (single arrowhead). In addition, autophosphorylation of an approximately 75 kDa protein of MELK was observed (Figure 4a; double arrowheads). Furthermore, we also confirmed that WT-MELK could phosphorylate GST-Bcl-GL but D150A-MELK could not (Figure 4b), indicating MELK can directly phosphorylate Bcl-GL. These findings suggest that Bcl-GL is a potential substrate for MELK.

Bottom Line: Northern blot analyses on multiple human tissues and cancer cell lines demonstrated that MELK was overexpressed at a significantly high level in a great majority of breast cancers and cell lines, but was not expressed in normal vital organs (heart, liver, lung and kidney).We also found that MELK physically interacted with Bcl-GL through its amino-terminal region.Our findings suggest that the kinase activity of MELK is likely to affect mammary carcinogenesis through inhibition of the pro-apoptotic function of Bcl-GL.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Molecular Medicine, Human Genome Center, Institute of Medical Science, The University of Tokyo, Tokyo, Japan.

ABSTRACT

Introduction: Cancer therapies directed at specific molecular targets in signaling pathways of cancer cells, such as tamoxifen, aromatase inhibitors and trastuzumab, have proven useful for treatment of advanced breast cancers. However, increased risk of endometrial cancer with long-term tamoxifen administration and of bone fracture due to osteoporosis in postmenopausal women undergoing aromatase inhibitor therapy are recognized side effects. These side effects as well as drug resistance make it necessary to search for novel molecular targets for drugs on the basis of well-characterized mechanisms of action.

Methods: Using accurate genome-wide expression profiles of breast cancers, we found maternal embryonic leucine-zipper kinase (MELK) to be significantly overexpressed in the great majority of breast cancer cells. To assess whether MELK has a role in mammary carcinogenesis, we knocked down the expression of endogenous MELK in breast cancer cell lines using mammalian vector-based RNA interference. Furthermore, we identified a long isoform of Bcl-G (Bcl-GL), a pro-apoptotic member of the Bcl-2 family, as a possible substrate for MELK by pull-down assay with recombinant wild-type and kinase-dead MELK. Finally, we performed TUNEL assays and FACS analysis, measuring proportions of apoptotic cells, to investigate whether MELK is involved in the apoptosis cascade through the Bcl-GL-related pathway.

Results: Northern blot analyses on multiple human tissues and cancer cell lines demonstrated that MELK was overexpressed at a significantly high level in a great majority of breast cancers and cell lines, but was not expressed in normal vital organs (heart, liver, lung and kidney). Suppression of MELK expression by small interfering RNA significantly inhibited growth of human breast cancer cells. We also found that MELK physically interacted with Bcl-GL through its amino-terminal region. Immunocomplex kinase assay showed that Bcl-GL was specifically phosphorylated by MELK in vitro. TUNEL assays and FACS analysis revealed that overexpression of wild-type MELK suppressed Bcl-GL-induced apoptosis, while that of D150A-MELK did not.

Conclusion: Our findings suggest that the kinase activity of MELK is likely to affect mammary carcinogenesis through inhibition of the pro-apoptotic function of Bcl-GL. The kinase activity of MELK could be a promising molecular target for development of therapy for patients with breast cancers.

Show MeSH
Related in: MedlinePlus