Browsing by Subject "MAPK"

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Heat shock-induced apoptosis
(2013-12) Mahajan, Indra Maria; Wright, Casey Wyatt; Bratton, Shawn B.
Apoptosis is a conserved program of cell death that promotes organism homeostasis in all stages of life. Two main pathways activate caspases, which are cysteinyl-aspartate proteases that execute apoptosis. The extrinsic pathway is initiated by cell surface death receptors, while the intrinsic pathway is initiated by intracellular signals that cause permeabilization of the outer mitochondrial membrane (MOMP). The Bcl-2 protein family regulates MOMP, which causes the release of several pro-apoptotic proteins (such as cytochrome c, Smac) into the cytosol. Bcl-2 proteins share homology in up to four "BH" domains and are subdivided into three subgroups. Pro-apoptotic Bax and Bak catalyze pore formation in the mitochondria, while anti-apoptotic members (Bcl-2, Mcl-1) inhibit MOMP. The third subgroup, termed BH3-only, promotes MOMP by either antagonizing Bcl-2 proteins or by directly activating Bax/Bak, and initiate apoptosis in response to various stressors, including heat shock (HS). Hyperthermia or acute HS reportedly induces apoptosis through caspase-2-mediated cleavage of BID, engaging the intrinsic pathway. However, additional evidence suggests that this pathway could represent an amplification loop. Thus we hypothesized that during HS, another BH3-only protein such as BIM, that does not require cleavage, could engage MOMP. Herein, we report that BIM mediates an alternative HS-induced apoptosis pathway. Cells lacking BIM are resistant to HS and exhibit better short and long-term survival than either Bid[superscript -/-] or Bax[superscript -/-]Bak[superscript -/-]. Moreover, caspase-2 induces apoptosis in Bim[superscript -/-] but not Bid[superscript -/-] cells, implying that caspase-2 kills exclusively through BID. Interestingly, Bim[superscript -/-] and Bax[superscript -/-]Bak[superscript -/-] cells are entirely resistant to MOMP, but the Bax[superscript -/-]Bak[superscript -/-] cells still undergo caspase-3 activation and remain partially sensitive to HS, indicating that BIM triggers caspase-3 activation upstream of mitochondria. Thus, BIM plays an important role in HS-induced apoptosis. Hyperthermia has clinical applications for the treatment of solid tumors. Unfortunately, a practical limitation is the development of thermotolerance, which confers resistance not only to subsequent HS but also to radiotherapy and chemotherapy. Therefore, a better understanding of the molecular mechanisms involved both in heat-induced apoptosis and thermotolerance could lead to new therapeutic interventions. Here we also show evidence for a putative role for the stress kinase JNK signaling pathway in the regulation of thermotolerance.
Investigation of c-Jun N-terminal Kinase 2 Regulation
(2017-04) Go, Jaeeun; Kaoud, Tamer; Dalby, Kevin
JNK2). This protein is unique in that it can autophosphorylate, or phosphorylate itself. It was found that these proteins tend to form aggregates of four, or tetramers, when inactive and remain as single units, or monomers, when active. In glioblastoma and lung carcinoma cells, JNK2s are reported to be constitutively, or always, active. This study aimed to investigate a selection of JNK2 mutations determine which of these mutations can alter JNK2 activity and oligomerization state. Results from size-exclusion chromatography and light scattering analysis (SEC-LS) suggested that while the wild type JNK2 exists as a mixture of monomers and tetramers, the activation loop chimera mutant (C177G/N179S) exists only as monomers. Additionally, phosphorylation assay analysis showed increased autophosphorylation activity of the activation loop chimera mutant compared to the wild type. These results suggest that the activation loop may be involved in the mechanism of JNK2 regulation. Elucidation of MAPK regulation mechanisms may allow more effective cancer therapy screening and increase overall understanding of tumor growth mechanisms.
Obesity enhances nongenomic estrogen receptor crosstalk with the PI3K/Akt and MAPK pathways to promote in vitro measures of breast cancer progression
(Breat Cancer Research, 2013-07-23) Bowers, Laura; Cavazos, David A.; Maximo, Ilane XF; Brenner, Andrew J.; Hursting, Srphen D.; deGraffenried, Linda A.
Introduction: Epidemiological and clinical studies indicate that obesity is associated with a worse postmenopausal breast cancer prognosis and an increased risk of endocrine therapy resistance. However, the mechanisms mediating these effects remain poorly understood. Here we investigate the molecular pathways by which obesity-associated circulating factors in the blood enhance estrogen receptor alpha (ERα) positive breast cancer cell viability and growth. Methods: Blood serum was collected from postmenopausal breast cancer patients and pooled by body mass index (BMI) category (Control: 18.5 to 24.9 kg/m2; Obese: ≥30.0 kg/m2). The effects of patient sera on MCF-7 and T47D breast cancer cell viability and growth were examined by MTT and colony formation assays, respectively. Insulin-like growth factor receptor 1(IGF-1R), Akt, and ERK1/2 activation and genomic ERα activity were assessed to determine their possible contribution to obese patient sera-induced cell viability and growth. To further define the relative contribution of these signaling pathways, cells grown in patient sera were treated with various combinations of ERα, PI3K/Akt and MAPK targeted therapies. Comparisons between cells exposed to different experimental conditions were made using one-way analysis of variance (ANOVA) and Student's t test. Results: Cells grown in media supplemented with obese patient sera displayed greater cell viability and growth as well as IGF-1R, Akt and ERK1/2 activation relative to control sera. Despite the lack of a significant difference in genomic ERα activity following growth in obese versus control patient sera, we observed a dramatic reduction in cell viability and growth after concurrent inhibition of the ERα and PI3K/Akt signaling pathways. Further, we demonstrated that ERα inhibition was sufficient to attenuate obese serum-induced Akt and ERK1/2 activation. Together, these data suggest that obesity promotes greater ERα positive breast cancer cell viability and growth through enhanced crosstalk between nongenomic ERα signaling and the PI3K/Akt and MAPK pathways. Conclusions: Circulating factors in the serum of obese postmenopausal women stimulate ERα positive breast cancer cell viability and growth by facilitating non-genomic ERα crosstalk with the PI3K/Akt and MAPK signaling pathways. These findings provide valuable insight into one mechanism by which obesity may promote ERα positive postmenopausal breast cancer progression and endocrine therapy resistance.
The Kinetic Mechanism of Multisite Phosphorylation of Activating Transcription Factor 2 by c-Jun N-terminal Kinase 2
(2024) DeJong, Audrey; Dalby, Kevin
The mitogen-activated protein kinases (MAPKs) signal transduction pathway has been well studied as a regulator of a wide range of cellular responses. Specifically, the three-kinase cascade that activates activating transcription factor 2 (ATF2) is implicated in cell proliferation, stress responses, and DNA damage repair. 1 While the kinetic mechanism of ATF2 phosphorylation by p38a has been shown to be non-processive, the phosphorylation mechanism of ATF2 by c-Jun N-terminal kinase 2 (JNK2) is currently unknown. 2 Here, we will investigate the steady-state kinetic mechanism of ATF2 phosphorylation by JNK2 by comparing its kinetic profile with a lower-affinity mutant JNK2 (R127A). Our results show that JNK2 (R127A) has a lower affinity for ATF2 (K m = 4.75 µM ± 0.92) with a similar catalytic efficiency (k cat /K m = 25 s -1 µM -1 ) to the wild-type JNK2 (k cat /K m = 20 s -1 µM -1 ). This data helps design future pre-steady-state kinetic experiments to investigate this mechanism further.