Substrate Specificity and Activity Regulation of Protein Kinase MELK

Monique Beullens,Sadia Vancauwenbergh,Nick Morrice,Rita Derua,Hugo Ceulemans,Etienne Waelkens,Mathieu Bollen

doi:10.1074/jbc.m507274200

Monique Beullens, Sadia Vancauwenbergh + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m507274200

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Abstract

Maternal embryonic leucine zipper kinase (MELK) is a protein Ser/Thr kinase that has been implicated in stem cell renewal, cell cycle progression, and pre-mRNA splicing, but its substrates and regulation are not yet known. We show here that MELK has a rather broad substrate specificity and does not appear to require a specific sequence surrounding its (auto)phosphorylation sites. We have mapped no less than 16 autophosphorylation sites including serines, threonines, and a tyrosine residue and show that the phosphorylation of Thr167 and Ser171 is required for the activation of MELK. The expression of MELK activity also requires reducing agents such as dithiothreitol or reduced glutathione. Furthermore, we show that MELK is a Ca2+-binding protein and is inhibited by physiological Ca2+ concentrations. The smallest MELK fragment that was still catalytically active comprises the N-terminal catalytic domain and the flanking ubiquitin-associated domain. A C-terminal fragment of MELK functions as an autoinhibitory domain. Our data show that the activity of MELK is regulated in a complex manner and offer new perspectives for the further elucidation of its biological function.

Highlights

The catalytic domain of the AMPK-related protein kinases is located in the N terminus of their catalytic subunit
Maternal embryonic leucine zipper kinase (MELK) phosphorylated a broad range of structurally unrelated proteins, such as myelin basic protein (MBP), Histone H1, and the splicing factors CDC5L, NIPP1, and SAP155
Our data indicate that MELK has a rather broad substrate specificity in vitro

Summary

Substrate Specificity and Activity Regulation of Protein Kinase MELK*

Maternal embryonic leucine zipper kinase (MELK) is a protein Ser/Thr kinase that has been implicated in stem cell renewal, cell cycle progression, and pre-mRNA splicing, but its substrates and regulation are not yet known. ZPR9 itself interacts with the transcription factor B-Myb, a regulator of cell proliferation and differentiation, and enhances its transcriptional activity Another interactor of MELK is the transcription and splicing factor NIPP1, but the binding of NIPP1 requires the phosphorylation of MELK on a specific threonine in its TP dipeptide-rich domain [14]. A major limitation in studying the role of MELK as a protein kinase is that it is not known what controls its activity and that MELK expressed in mammalian cells seems to be inactive [5]. We show here that MELK has a rather broad substrate specificity and that its activity is complexly regulated by autophosphorylation, autoinhibition, Ca2ϩ ions, and reducing agents

EXPERIMENTAL PROCEDURES

Substrate Specificity and Regulation of MELK

RESULTS

Phosphorylated residue

DISCUSSION