Cooperation of ERK and SCFSkp2 for MKP-1 destruction provides a positive feedback regulation of proliferating signaling.
The dual-specificity MAPK phosphatase MKP-1/CL100/DUSP1 is an inducible nuclear protein controlled by p44/42 MAPK (ERK1/2) in a negative feedback mechanism to inhibit kinase activity. Here, we report on the molecular basis for a novel positive feedback mechanism to sustain ERK activation by triggering MKP-1 proteolysis. Active ERK2 docking to the DEF motif (FXFP, residues 339-342) of N-terminally truncated MKP-1 in vitro initiated phosphorylation at the Ser(296)/Ser(323) domain, which was not affected by substituting Ala for Ser at Ser(359)/Ser(364). The DEF and Ser(296)/Ser(323) sites were essential for ubiquitin-mediated MKP-1 proteolysis stimulated by MKK1-ERK signaling in H293 cells, whereas the N-terminal domain and Ser(359)/Ser(364) sites were dispensable. ERK activation by serum increased the endogenous level of ubiquitinated phospho-Ser(296) MKP-1 and the degradation of MKP-1. Intriguingly, active ERK-promoted phospho-Ser(296) MKP-1 bound to SCF(Skp2) ubiquitin ligase in vivo and in vitro. Forced expression of Skp2 enhanced MKP-1 polyubiquitination and proteolysis upon ERK activation, whereas depletion of endogenous Skp2 suppressed such events. The kinetics of ERK signaling stimulated by serum correlated with the endogenous MKP-1 degradation rate in a Skp2-dependent manner. Thus, MKP-1 proteolysis can be achieved via ERK and SCF(Skp2) cooperation, thereby sustaining ERK activation.
Lin
Yun-Wei
YW
Molecular Carcinogenesis Laboratory, Institute of Biotechnology, Hsinchu, Taiwan.
Yang
Jia-Ling
JL
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001665:Binding Sites; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D049109:Cell Proliferation; D002471:Cell Transformation, Neoplastic; D004305:Dose-Response Relationship, Drug; D054638:Dual Specificity Phosphatase 1; D004789:Enzyme Activation; D004926:Escherichia coli / Q000378:metabolism; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D025461:Feedback, Physiological; D015971:Gene Expression Regulation, Enzymologic; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D017874:Immediate-Early Proteins / Q000378:metabolism*; D007700:Kinetics; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D008954:Models, Biological; D008957:Models, Genetic; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D054645:Protein Phosphatase 1; D017434:Protein Structure, Tertiary; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism*; D011506:Proteins / Q000737:chemistry; D034741:RNA, Small Interfering / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D044786:S-Phase Kinase-Associated Proteins / Q000378:metabolism*; D044843:SKP Cullin F-Box Protein Ligases / Q000378:metabolism*; D012694:Serine / Q000737:chemistry; D015398:Signal Transduction; D013997:Time Factors; D014162:Transfection; D025801:Ubiquitin / Q000737:chemistry
D018797:Cell Cycle Proteins; D017874:Immediate-Early Proteins; D011506:Proteins; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; D044786:S-Phase Kinase-Associated Proteins; D025801:Ubiquitin; D012694:Serine; D044843:SKP Cullin F-Box Protein Ligases; D005982:Glutathione Transferase; D048049:Extracellular Signal-Regulated MAP Kinases; D019950:Mitogen-Activated Protein Kinase 1; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; C517019:DUSP1 protein, human; D054638:Dual Specificity Phosphatase 1; D017027:Protein Tyrosine Phosphatases