Jiing-Dwan Lee

Characterization of the structure and function of a novel MAP kinase kinase (MKK6)

Mitogen-activated protein (MAP) kinases require dual phosphorylation on threonine and tyrosine residues in order to gain enzymatic activity. This activation is carried out by a family of enzymes known as MAP kinase kinases (MKKs or MEKs). It appears that there are at least four subgroups in this family; MEK1/MEK2 subgroup that activates ERK1/ERK2, MEK5 that activates ERK5/BMK1, MKK3 that activates p38, and MKK4 that activates p38 and Jun kinase. Here we describe the characteristics of a new MKK termed MKK6. The clones we isolated encode two splice isoforms of human MKK6 comprised of 278 and 334 amino acids, respectively, and one murine MKK6 with 237 amino acids. Sequence information derived from cDNA cloning indicated that MKK6 is most closely related to MKK3. The functional data revealed from co-transfection assays suggests that MKK6, like MKK3, selectively phosphorylates p38. Unlike the previously described MKKs (or MEKs), MKK6 exists in a variety of alternatively spliced isoforms with distinct patterns of tissue expression. This suggests novel mechanisms regulating activation and/or function of various forms of MKK6.

Characterization of a selective inhibitor of the Parkinson's disease kinase LRRK2

Mutations in leucine-rich repeat kinase 2 (LRRK2) are strongly associated with late-onset autosomal dominant Parkinson’s disease. We employed a novel, parallel, compound-centric approach to identify a potent and selective LRRK2 inhibitor LRRK2-IN-1, and demonstrated that inhibition of LRRK2 induces dephosphorylation of Ser910/Ser935 and accumulation of LRRK2 within aggregate structures. LRRK2-IN-1 will serve as a versatile tool to pharmacologically interrogate LRRK2 biology and study its role in Parkinson’s disease.

Role of the BMK1/ERK5 signaling pathway: lessons from knockout mice.

Mitogen-activated protein (MAP) kinase cascades play a central role in mediating extracellular stimuli-induced intracellular signaling during cell activation. The fourth and least studied mammalian MAP kinase pathway, big MAP kinase 1 (BMK1), also known as extracellular signal regulated kinase 5 (ERK5), is activated in response to growth factors and stress. Activation of this signaling pathway has been implicated not only in physiological functions such as cell survival, proliferation and differentiation but also in pathological processes such as carcinogenesis, cardiac hypertrophy and atherosclerosis. In recent years a series of gene-targeted mice lacking components within the BMK1 cascade have been generated, which have enabled us to investigate the role of the BMK1 pathway within different tissues. Analyses of these knockout mice have led to major discoveries in the role of BMK1 signaling in angiogenesis and in cardiac development. Moreover, studies using conditional BMK1 knockout mice, which circumvent the early embryonic lethality of BMK1 knockouts, have unveiled the importance of BMK1 in endothelial survival and maintenance of vascular integrity during adulthood. Here we summarize current understanding of the function of BMK1, as well as include new data generated from a series of tissue-specific BMK1 knockout mice in an attempt to dissect the role of the BMK1 pathway in various cell types in animals.

CDC25B Mediates Rapamycin-Induced Oncogenic Responses in Cancer Cells

Because the mammalian target of rapamycin (mTOR) pathway is commonly deregulated in human cancer, mTOR inhibitors, rapamycin and its derivatives, are being actively tested in cancer clinical trials. Clinical updates indicate that the anticancer effect of these drugs is limited, perhaps due to rapamycin-dependent induction of oncogenic cascades by an as yet unclear mechanism. As such, we investigated rapamycin-dependent phosphoproteomics and discovered that 250 phosphosites in 161 cellular proteins were sensitive to rapamycin. Among these, rapamycin regulated four kinases and four phosphatases. A siRNA-dependent screen of these proteins showed that AKT induction by rapamycin was attenuated by depleting cellular CDC25B phosphatase. Rapamycin induces the phosphorylation of CDC25B at Serine375, and mutating this site to Alanine substantially reduced CDC25B phosphatase activity. Additionally, expression of CDC25B (S375A) inhibited the AKT activation by rapamycin, indicating that phosphorylation of CDC25B is critical for CDC25B activity and its ability to transduce rapamycin-induced oncogenic AKT activity. Importantly, we also found that CDC25B depletion in various cancer cell lines enhanced the anticancer effect of rapamycin. Together, using rapamycin phosphoproteomics, we not only advance the global mechanistic understanding of the action of rapamycin but also show that CDC25B may serve as a drug target for improving mTOR-targeted cancer therapies.

Big Mitogen-Activated Protein Kinase 1/Extracellular Signal-Regulated Kinase 5 Signaling Pathway Is Essential for Tumor-Associated Angiogenesis

Although big mitogen-activated protein kinase 1 (BMK1) has been shown to be critical for embryonic angiogenesis, the role of BMK1 in tumor-associated neovascularization is poorly understood. Exogenous tumors were established in BMK1+/+, BMK1flox/+, or BMK1flox/flox mice carrying the Mx1-Cre transgene. Induced deletion of host BMK1 gene significantly reduced the volumes of B16F10 and LL/2 tumor xenografts in BMK1flox/flox mice by 63% and 72%, respectively. Examining the tumors in these induced BMK1-knockout animals showed a significant decrease in vascular density. Localized reexpression of BMK1 in BMK1-knockout mice by administration of adenovirus encoding BMK1 restored tumor growth and angiogenesis to the levels observed in wild-type mice. These observations were further supported by in vivo Matrigel plug assays in which vascular endothelial growth factor- and basic fibroblast growth factor-induced neovacularization was impaired by removing BMK1. Through screening with the Pepchip microarray, we discovered that in BMK1-knockout endothelial cells, phosphorylation of ribosomal protein S6 (rpS6) at Ser235/236 was mostly abrogated, and this BMK1-dependent phosphorylation required the activity of p90 ribosomal S6 kinase (RSK). Immunofluorescent analysis of tumor vasculature from BMK1-knockout and control animals revealed a strong correlation between the presence of BMK1 and the phosphorylation of rpS6 in tumor-associated endothelial cells of blood vessels. As both RSK and rpS6 are known to be important for cell proliferation and survival, which are critical endothelial cell functions during neovascularization, these findings suggest that the BMK1 pathway is crucial for tumor-associated angiogenesis through its role in the regulation of the RSK-rpS6 signaling module.

Combined Integrin Phosphoproteomic Analyses and Small Interfering RNA–Based Functional Screening Identify Key Regulators for Cancer Cell Adhesion and Migration

Integrins interact with extracellular matrix (ECM) and deliver intracellular signaling for cell proliferation, survival, and motility. During tumor metastasis, integrin-mediated cell adhesion to and migration on the ECM proteins are required for cancer cell survival and adaptation to the new microenvironment. Using stable isotope labeling by amino acids in cell culture-mass spectrometry, we profiled the phosphoproteomic changes induced by the interactions of cell integrins with type I collagen, the most common ECM substratum. Integrin-ECM interactions modulate phosphorylation of 517 serine, threonine, or tyrosine residues in 513 peptides, corresponding to 357 proteins. Among these proteins, 33 key signaling mediators with kinase or phosphatase activity were subjected to small interfering RNA-based functional screening. Three integrin-regulated kinases, DBF4, PAK2, and GRK6, were identified for their critical role in cell adhesion and migration possibly through their regulation of actin cytoskeleton arrangement. Altogether, we not only depict an integrin-modulated phosphorylation network during cell-ECM protein interactions but also reveal novel regulators for cell adhesion and migration.

A crucial role of mitochondrial Hsp40 in preventing dilated cardiomyopathy

Many heat-shock proteins (Hsp) are members of evolutionarily conserved families of chaperone proteins that inhibit the aggregation of unfolded polypeptides and refold denatured proteins, thereby remedying phenotypic effects that may result from protein aggregation or protein instability. Here we report that the mitochondrial chaperone Hsp40, also known as Dnaja3 or Tid1, is differentially expressed during cardiac development and pathological hypertrophy. Mice deficient in Dnaja3 developed dilated cardiomyopathy (DCM) and died before 10 weeks of age. Progressive respiratory chain deficiency and decreased copy number of mitochondrial DNA were evident in cardiomyocytes lacking Dnaja3. Profiling of Dnaja3-interacting proteins identified the α-subunit of DNA polymerase γ (Polga) as a client protein. These findings suggest that Dnaja3 is crucial for mitochondrial biogenesis, at least in part, through its chaperone activity on Polga and provide genetic evidence of the necessity for mitochondrial Hsp40 in preventing DCM.

Protein tyrosine phosphatase PTPN13 negatively regulates Her2/ErbB2 malignant signaling.

Deregulated Her2/ErbB2 receptor tyrosine kinase drives tumorigenesis and tumor progression in a variety of human tissues. Her2 transmits oncogenic signals through phosphorylation of its cytosolic domain. To study innate cellular mechanisms for containing Her2 oncogenic phosphorylation, a siRNA phosphatase library was screened for cellular phosphatase(s) that enhance phosphorylation in the signaling motif of Her2 after knockdown. We found that silencing protein tyrosine phosphatase PTPN13 significantly augmented growth factor-induced phosphorylation of the Her2 signaling domain and promoted the invasiveness of Her2-deregulated cancer cells. In addition, we discovered that growth factor-induced phosphorylation of PTPN13 was essential for the dephosphorylation of Her2 suggesting a negative feedback mechanism induced by growth factor to inhibit cellular Her2 activity through PTPN13. Importantly, we showed that PTPN13 mutations previously reported in human tumors significantly reduced the phosphatase activity of PTPN13, and consequently elevated the oncogenic potential of Her2 and the invasiveness of Her2-overexpressing human cancer cells. Taken together, these results suggest that cellular PTPN13 inhibits Her2 activity by dephosphorylating the signal domain of Her2 and plays a role in attenuating invasiveness and metastasis of Her2 overactive tumors.

Role of BMK1 in regulation of growth factor-induced cellular responses

Big mitogen-activated protein kinase (MAPK) 1 (BMK1), also known as ERK5, is a recently identified member of the mammalian MAPK family. Cellular stimulation of BMK1 is induced in response to growth factors, oxidative stress, and hyperosmolar conditions. Specific members of the myocyte enhancer factor 2 family of transcription factors that regulate growth factor-induced early gene expression have been identified as direct downstream targets of BMK1 activity. Recent studies have shown that growth factors of the epidermal growth factor family mediate the sequential activation of a kinase cascade consisting of MAPK kinase kinase 3, MAPK kinase 5, and BMK1. Most importantly, the activation of this signal transduction pathway has been shown to be required for growth factor-mediated cell proliferation and cell-cycle progression. Collectively, these studies establish BMK1 as an important regulator of growth factor-induced cellular responses.

Tid1, the Human Homologue of a Drosophila Tumor Suppressor, Reduces the Malignant Activity of ErbB-2 in Carcinoma Cells

The ErbB-2/HER-2 receptor tyrosine kinase is overexpressed in a wide range of solid human tumors. The ErbB-2 gene product is a transmembrane glycoprotein belonging to the epidermal growth factor receptor family, and its cytoplasmic domain is responsible for sending the mitogenic signals into cells. We discovered that this domain of ErbB-2 interacts with Tid1 protein, the human counterpart of the Drosophila tumor suppressor Tid56, whose null mutation causes lethal tumorigenesis during the larval stage. Tid1 also is known as a cochaperone of heat shock protein 70 (HSP70) and binds to HSP70 through its conserved DnaJ domain. We found that increased expression of Tid1 in human mammary carcinomas overexpressing ErbB-2 suppresses the expression level of ErbB-2 and attenuates the resultant ErbB-2–dependent oncogenic extracellular signal-regulated kinase 1/2 and big mitogen-activated protein kinase 1 signaling pathways leading to programmed cell death (PCD). A functional DnaJ domain of Tid1 also is required for its inhibition of ErbB-2 expression and the consequent PCD of carcinoma cells resulting from increased Tid1 expression. Importantly, ErbB-2–dependent tumor progression in animals is inhibited by increased expression of Tid1 in tumor cells. Collectively, these results suggest that Tid1 modulates the uncontrolled proliferation of ErbB-2–overexpressing carcinoma cells by reducing ErbB-2 expression and as a result suppresses the ErbB-2–dependent cancerous signaling and tumor progression. Moreover, the cochaperonic and regulatory functions of Tid1 on HSP70 most likely play an essential role in this antitumor function of Tid1 in carcinoma cells.