Induced, selective proteolysis of MLK3 negatively regulates MLK3/JNK signalling

Geou Yarh Liou, Hua Zhang, Eva M. Miller, Steve A. Seibold, Weiqin Chen, Kathleen A. Gallo

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

MLK3 (mixed lineage kinase 3) is a MAP3K [MAPK (mitogen-activated protein kinase) kinase kinase] that activates multiple MAPK pathways, including the JNK (c-Jun N-terminal kinase) pathway. Immunoblotting of lysates from cells ectopically expressing active MLK3 revealed an additional immunoreactive band corresponding to a CTF (C-terminal fragment) of MLK3. In the present paper we provide evidence that MLK3 undergoes proteolysis to generate a stable CTF in response to different stimuli, including PMA and TNFα (tumour necrosis factor α). The cleavage site was deduced by Edman sequencing as between Gln251 and Pro252, which is within the kinase domain of MLK3. Based on our homology model of the kinase domain of MLK3, the region containing the cleavage site is predicted to reside on a flexible solvent-accessible loop. Site-directed mutagenesis studies revealed that Leu250 and Gln251 are required for recognition by the 'MLK3 protease', reminiscent of the substrate specificity of the coronavirus 3C and 3CL proteases. Whereas numerous mammalian protease inhibitors have no effect on MLK3 proteolysis, blockade of the proteasome through epoxomicin or MG132 abolishes PMA-induced production of the CTF of MLK3. This CTF is able to heterodimerize with full-length MLK3, and interact with the active form of the small GTPase Cdc42, resulting in diminished activation loop phosphorylation of MLK3 and reduced signalling to JNK. Thus this novel proteolytic processing of MLK3 may negatively control MLK3 signalling to JNK.

Original languageEnglish (US)
Pages (from-to)435-443
Number of pages9
JournalBiochemical Journal
Volume427
Issue number3
DOIs
StatePublished - May 1 2010
Externally publishedYes

Fingerprint

Proteolysis
JNK Mitogen-Activated Protein Kinases
mitogen-activated protein kinase kinase kinase 11
Peptide Hydrolases
Phosphotransferases
MAP Kinase Kinase Kinases
Mutagenesis
Coronavirus
Phosphorylation
Monomeric GTP-Binding Proteins
Proteasome Endopeptidase Complex
Substrate Specificity
Site-Directed Mutagenesis
Protease Inhibitors
Immunoblotting

Keywords

  • c-Jun N-terminal kinase (JNK)
  • Epoxomicin
  • Mitogen-activated protein kinase (MAPK)
  • Mixed lineage kinase 3 (MLK3)
  • Proteasome
  • Tumour necrosis factor α (TNFα)

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Liou, G. Y., Zhang, H., Miller, E. M., Seibold, S. A., Chen, W., & Gallo, K. A. (2010). Induced, selective proteolysis of MLK3 negatively regulates MLK3/JNK signalling. Biochemical Journal, 427(3), 435-443. https://doi.org/10.1042/BJ20091077

Induced, selective proteolysis of MLK3 negatively regulates MLK3/JNK signalling. / Liou, Geou Yarh; Zhang, Hua; Miller, Eva M.; Seibold, Steve A.; Chen, Weiqin; Gallo, Kathleen A.

In: Biochemical Journal, Vol. 427, No. 3, 01.05.2010, p. 435-443.

Research output: Contribution to journalArticle

Liou, GY, Zhang, H, Miller, EM, Seibold, SA, Chen, W & Gallo, KA 2010, 'Induced, selective proteolysis of MLK3 negatively regulates MLK3/JNK signalling', Biochemical Journal, vol. 427, no. 3, pp. 435-443. https://doi.org/10.1042/BJ20091077
Liou, Geou Yarh ; Zhang, Hua ; Miller, Eva M. ; Seibold, Steve A. ; Chen, Weiqin ; Gallo, Kathleen A. / Induced, selective proteolysis of MLK3 negatively regulates MLK3/JNK signalling. In: Biochemical Journal. 2010 ; Vol. 427, No. 3. pp. 435-443.
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