Dynamic Positive Feedback Phosphorylation of Mixed Lineage Kinase 3 by JNK Reversibly Regulates Its Distribution to Triton-soluble Domains

Karen A Schachter,Yan Du,Anning Lin,Kathleen A Gallo

doi:10.1074/jbc.m603324200

Karen A Schachter, Yan Du + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m603324200

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Abstract

MLK3 (mixed lineage kinase 3) is a widely expressed, mammalian serine/threonine protein kinase that activates multiple MAPK pathways. Previously our laboratory used in vivo labeling/mass spectrometry to identify phosphorylation sites of activated MLK3. Seven of 11 identified sites correspond to the consensus motif for phosphorylation by proline-directed kinases. Based on these results, we hypothesized that JNK, or another proline-directed kinase, phosphorylates MLK3 as part of a feedback loop. Herein we provide evidence that MLK3 can be phosphorylated by JNK in vitro and in vivo. Blockade of JNK results in dephosphorylation of MLK3. The hypophosphorylated form of MLK3 is inactive and redistributes to a Triton-insoluble fraction. Recovery from JNK inhibition restores MLK3 solubility and activity, indicating that the redistribution process is reversible. This work describes a novel mode of regulation of MLK3, by which JNK-mediated feedback phosphorylation of MLK3 regulates its activation and deactivation states by cycling between Triton-soluble and Triton-insoluble forms.

Highlights

MLK3 is a widely expressed mammalian serine/threonine kinase that functions as a mitogenactivated protein kinase kinase kinase (MAPKKK)2 and can activate multiple MAPK pathways [1]
JNK Is Required for in Vivo Phosphorylation of MLK3—Using mass spectrometry coupled with comparative phosphotryptic peptide mapping, we previously identified 11 in vivo phosphorylation sites on MLK3 [25]
After affinity purification and trypsin digestion of MLK3, the resultant radiolabeled phosphotryptic peptides were resolved by chromatography, and their sequences were determined using a combination of matrix-assisted laser desorption ionization mass spectrometry and liquid chromatography-tandem mass spectrometry

Summary

Introduction

MLK3 (mixed lineage kinase 3) is a widely expressed mammalian serine/threonine kinase that functions as a mitogenactivated protein kinase kinase kinase (MAPKKK)2 and can activate multiple MAPK pathways [1]. To determine whether selective activation of JNK promotes MLK3 phosphorylation, HeLa cells were transfected with expression vectors for MLK3 and the fusion protein JNKK2-JNK1. The amount of MLK3 in cellular lysates (Fig. 2B) increases when the corresponding cells express the constitutively active JNK fusion protein, whereas the levels of green fluorescent protein used as a transfection control remain constant.

Results

Conclusion