Regulation of Drosophila MKP-3 by Drosophila ERK.
DMKP-3 is a Drosophila dual-specificity phosphatase, which has high substrate specificity for Drosophila extracellular signal-regulated kinases (DERK). By in vitro reconstitution experiments, we found that DERK activates DMKP-3. Moreover, DMKP-3 was specifically activated by the addition of DERK but not by DJNK, Dp38, or Sevenmaker DERK D334N, a DMKP-3- binding mutant. The phosphatase activity of DMKP-3-R56A/R57A, a DERK-binding mutant, was not increased by DERK. Significantly, mammalian MKP-3 was also found to be activated by DERK. This cross-reactivity suggests a high level of conservation of the activation mechanism of ERK-specific phosphatases in Drosophila and mammals. When DMKP-3 was co-expressed with DERK in Drosophila Schneider cells, DMKP-3 protein levels increased, but this was not observed for the co-expressions of DJNK or Dp38. The stabilizations of the DERK binding mutants (DMKP-3-RR and DMKP-3-CA-RR) were not increased by DERK co-expression. Our results suggest that DERK specifically regulates DMKP-3 in terms of its enzyme activity and protein stability, and that direct protein-protein interaction is an essential aspect of this regulation.
Kim
Sung-Eun
SE
Department of Biotechnology, Protein Research Center, Yonsei University College of Engineering, Seoul 120-752, South Korea.
Kim
Sun-Hong
SH
Choi
Kang-Yell
KY
D000818:Animals; D002460:Cell Line; D017931:DNA Primers; D004330:Drosophila / Q000166:cytology / Q000235:genetics* / Q000502:physiology; D029721:Drosophila Proteins / Q000235:genetics* / Q000378:metabolism; D054642:Dual Specificity Phosphatase 6; D005982:Glutathione Transferase; D007700:Kinetics; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D010957:Plasmids; D016133:Polymerase Chain Reaction; D017027:Protein Tyrosine Phosphatases / Q000235:genetics* / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D017931:DNA Primers; D029721:Drosophila Proteins; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D011505:Protein-Tyrosine Kinases; D020928:Mitogen-Activated Protein Kinases; D054642:Dual Specificity Phosphatase 6; C486844:Mkp3 protein, Drosophila; D017027:Protein Tyrosine Phosphatases