Dataset Information

Context-dependent regulatory mechanisms control site-specific information processing within the MEK/ERK module

ABSTRACT: The impact of the cellular context and receptor type on the dynamics of phosphorylation cycles in signaling pathways are unclear. We employ an unbiased approach to identify network alterations that explain phosphorylation dynamics of the MEK/ERK module in response to hepatocyte growth factor or interleukin-6, comparing primary hepatocytes with the immortalized cell line HaCaT. By combining quantitative mass spectrometry with dynamic modeling, we elucidate the network structure in primary hepatocytes. For HaCaT, our model predicts additional dual feedback regulation, which we experimentally identify as DUSP6 and paxillin. Model analyses reveal switch-like ERK phospho-form distribution profiles in primary hepatocytes that are severely altered in HaCaT, explaining threshold, sensitivity and saturation behavior. We apply our approach to human hepatocellular carcinoma samples and identify, as predicted, elevated threonine-phosphorylated ERK levels in the tumor. This suggests that the prevalence of the mono-phosphorylated ERK rather than the doubly phosphorylated ERK is indicative for dysregulated proliferation.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Primary Cell, Hepatocyte, Liver, Cell Culture, Keratinocyte

DISEASE(S): Hepatocellular Carcinoma

SUBMITTER: Alexander Held

LAB HEAD: Marcel Schilling

PROVIDER: PXD002460 | Pride | 2016-02-08

REPOSITORIES: Pride

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Context-specific flow through the MEK/ERK module produces cell- and ligand-specific patterns of ERK single and double phosphorylation.

Iwamoto Nao N D'Alessandro Lorenza A LA Depner Sofia S Hahn Bettina B Kramer Bernhard A BA Lucarelli Philippe P Vlasov Artyom A Stepath Markus M Böhm Martin E ME Deharde Daniela D Damm Georg G Seehofer Daniel D Lehmann Wolf D WD Klingmüller Ursula U Schilling Marcel M

Science signaling 20160202 413

The same pathway, such as the mitogen-activated protein kinase (MAPK) pathway, can produce different cellular responses, depending on stimulus or cell type. We examined the phosphorylation dynamics of the MAPK kinase MEK and its targets extracellular signal-regulated kinase 1 and 2 (ERK1/2) in primary hepatocytes and the transformed keratinocyte cell line HaCaT A5 exposed to either hepatocyte growth factor or interleukin-6. By combining quantitative mass spectrometry with dynamic modeling, we el ...[more]

PMID: 26838549

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Project description:The extracellular signal-regulated kinases (Erks), Erk1 and Erk2, are crucial components of the receptor tyrosine kinase (RTK)-Ras-Raf-MEK pathway, which is frequently mutated and constitutively activated in various cancers. Despite Erk activity being commonly observed in cancer, the role of direct Erk activation in driving tumorigenesis remains unproven. This study explores the oncogenic potential of intrinsically active Erk1 mutants, particularly Erk1R84H, in both in vivo and in vitro models. We established a transgenic mouse model enabling tissue-specific and temporally controlled expression of Erk1R84H. Upon inducing Erk1R84H expression in the liver, we observed the development of hepatocellular carcinoma (HCC) characterized by increased liver weight, necrosis, and significant loss of body weight. Intriguingly, the phosphorylated/active form of Erk1R84H was dramatically downregulated during HCC development, becoming almost undetectable in mature tumors. This phenomenon was mirrored in NIH3T3 cells transformed by Erk1R84H, where TEY phosphorylation was significantly reduced as transformed foci appeared. Our proteomic and phosphoproteomic analyses of stable clones expressing Erk1R84H or Erk1R84S revealed substantial changes in protein expression and kinase activity. Key findings included the upregulation of CDK1, AURKB, and CDK2, and the downregulation of DNA repair and circadian rhythm-related kinases. Notably, Upp1, a crucial enzyme in nucleotide metabolism, was highly upregulated in clones transformed by Erk1R84S or Erk1R84H, suggesting its role in supporting the transformed phenotype. Furthermore, we demonstrated that the feedback inhibition mechanism suppressing Erk phosphorylation is dependent on Erk activity itself. Inhibition of Erk1R84H or Erk1R84S with Erk inhibitors restored TEY phosphorylation, highlighting the importance of precise rather very low Erk activity in maintaining the transformed state. In summary, Erk1R84H is confirmed as an efficient oncoprotein capable of inducing HCC in vivo and transforming cells in culture. The mechanism it uses for transformation, involving significant down regulation of its own activity underscores the notion that too high oncogenic activity is not favored by the tumor and proposes a modified therapeutic approach of targeting Erk for its activation rather than inhibition.

Project description:The extracellular signal-regulated kinases (Erks), Erk1 and Erk2, are crucial components of the receptor tyrosine kinase (RTK)-Ras-Raf-MEK pathway, which is frequently mutated and constitutively activated in various cancers. Despite Erk activity being commonly observed in cancer, the role of direct Erk activation in driving tumorigenesis remains unproven. This study explores the oncogenic potential of intrinsically active Erk1 mutants, particularly Erk1R84H, in both in vivo and in vitro models.We established a transgenic mouse model enabling tissue-specific and temporally controlled expression of Erk1R84H. Upon inducing Erk1R84H expression in the liver, we observed the development of hepatocellular carcinoma (HCC) characterized by increased liver weight, necrosis, and significant loss of body weight. Intriguingly, the phosphorylated/active form of Erk1R84H was dramatically downregulated during HCC development, becoming almost undetectable in mature tumors. This phenomenon was mirrored in NIH3T3 cells transformed by Erk1R84H, where TEY phosphorylation was significantly reduced as transformed foci appeared.Our proteomic and phosphoproteomic analyses of stable clones expressing Erk1R84H or Erk1R84S revealed substantial changes in protein expression and kinase activity. Key findings included the upregulation of CDK1, AURKB, and CDK2, and the downregulation of DNA repair and circadian rhythm-related kinases. Notably, Upp1, a crucial enzyme in nucleotide metabolism, was highly upregulated in clones transformed by Erk1R84S or Erk1R84H, suggesting its role in supporting the transformed phenotype.Furthermore, we demonstrated that the feedback inhibition mechanism suppressing Erk phosphorylation is dependent on Erk activity itself. Inhibition of Erk1R84H or Erk1R84S with Erk inhibitors restored TEY phosphorylation, highlighting the importance of precise rather very low Erk activity in maintaining the transformed state.In summary, Erk1R84H is confirmed as an efficient oncoprotein capable of inducing HCC in vivo and transforming cells in culture. The mechanism it uses for transformation, involving significant down regulation of its own activity underscores the notion that too high oncogenic activity is not favored by the tumor and proposes a modified therapeutic approach of targeting Erk for its activation rather than inhibition.

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