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Molecular cloning, expression, and characterization of the human mitogen-activated protein kinase p44erk1
D L Charest1, G Mordret, K W Harder
1Biomedical Research Centre, University of British Columbia, Vancouver, Canada.
Abstract:
p44erk1 is a member of a family of tyrosyl-phosphorylated and mitogen-activated protein (MAP) kinases that participate in cell cycle control. A full-length erk1 cDNA was isolated from a human hepatoma cell line (Hep G2) library. The erk1 cDNA clone shared approximately 96% predicted amino acid identity with partial sequences of rodent erk1 cognates, and the erk1 gene was assigned to human chromosome 16 by hybrid panel analysis. Human erk1 expressed in Escherichia coli as a glutathione S-transferase fusion (GST-Erk1) protein was substantially phosphorylated on tyrosine in vivo. It underwent further autophosphorylation in vitro (up to 0.01 mol of P per mol) at the regulatory Tyr-204 site and at additional tyrosine and serine residues. Threonine autophosphorylation, presumably at the regulatory Thr-202 site, was also detected weakly when the recombinant kinase was incubated in the presence of manganese, but not in the presence of magnesium. Before and after cleavage of the GST-Erk1 protein with thrombin, it exhibited a relatively high level of myelin basic protein phosphotransferase activity, which could be reduced eightfold by treatment of the kinase with the protein-tyrosine phosphatase CD45, but not by treatment with the protein-serine/threonine phosphatase 2A. The protein-tyrosine kinase p56lck catalyzed phosphorylation of GST-Erk1 at two autophosphorylations sites, including Tyr-204, and at a novel site. A further fivefold stimulation of the myelin basic protein phosphotransferase activity of the GST-Erk1 was achieved in the presence of a partially purified MAP kinase kinase from sheep platelets. Under these circumstances, there was primarily an enhancement of the tyrosine phosphorylation of GST-Erk1. This MAP kinase kinase also similarly phosphorylated a catalytically compromised version of GST-Erk1 in which Lys-71 was converted to Ala by site-directed mutagenesis.
Insights
Human ERK1, a mitogen-activated protein kinase involved in cell cycle control, was cloned and characterized. Its enzymatic activity was modulated by phosphorylation and other kinases, highlighting its role in cellular signaling pathways.
Area of Science:
- Molecular Biology
- Biochemistry
- Cell Biology
Background:
- ERK1 (Extracellular signal-regulated kinase 1) is a key member of the mitogen-activated protein kinase (MAPK) family.
- MAP kinases are crucial regulators of cell cycle control and other cellular processes.
- Understanding ERK1's function requires detailed characterization of its structure, phosphorylation, and enzymatic activity.
Purpose of the Study:
- To isolate and characterize the full-length human ERK1 cDNA.
- To investigate the phosphorylation status and enzymatic activity of recombinant human ERK1.
- To explore the regulation of ERK1 activity by other kinases and phosphatases.
Main Methods:
- Cloning of full-length human ERK1 cDNA from a Hep G2 cell line library.
- Expression of human ERK1 in E. coli as a glutathione S-transferase (GST-Erk1) fusion protein.
- In vitro and in vivo phosphorylation assays, including autophosphorylation and phosphorylation by other kinases (p56lck, MAP kinase kinase).
- Enzymatic activity assays using myelin basic protein as a substrate.
- Treatment with protein-tyrosine phosphatase CD45 and protein-serine/threonine phosphatase 2A to assess dephosphorylation effects.
- Site-directed mutagenesis to create a catalytically compromised ERK1 mutant.
Main Results:
- Human ERK1 cDNA was successfully isolated and sequenced, showing high homology to rodent cognates.
- The ERK1 gene was mapped to human chromosome 16.
- Recombinant GST-Erk1 protein exhibited significant tyrosine phosphorylation in vivo and autophosphorylation in vitro at specific regulatory sites (Tyr-204, Thr-202).
- GST-Erk1 displayed myelin basic protein phosphotransferase activity, which was reduced by CD45 but not by PP2A.
- The protein-tyrosine kinase p56lck phosphorylated GST-Erk1 at known and novel sites.
- MAP kinase kinase from sheep platelets stimulated GST-Erk1 activity and tyrosine phosphorylation, and also phosphorylated a catalytically inactive ERK1 mutant.
Conclusions:
- Human ERK1 is a conserved protein kinase involved in cellular signaling.
- ERK1 activity is regulated by complex phosphorylation events, including autophosphorylation and cross-regulation by other kinases.
- The findings provide insights into the molecular mechanisms governing ERK1 activation and function in cell cycle control.