Complete Nucleotide Sequence, Expression, and Chromosomal Localisation of Human Mixed‐Lineage Kinase 2

Abstract

Protein kinases play pivotal roles in the control of many cellular processes. In a search for protein kinases expressed in human epithelial tumour cells, we discovered two members of a novel protein kinase family [Dorow. D. S., Devereux, L., Dietzsch, E. & de Kretser, T. A. (1993) Eul: J. Biochem. 213, 701 -7101. Due to the unique mixture of structural domains within their amino acid sequences, we named the family mixed-lineage kinases (MLK). We initially isolated clones encoding partial cDNAs of MLKl and 2 from a human colonic cDNA library. The MLK2 cDNA was subsequently used to screen a human brain cDNA library and we have now cloned and sequenced a 3454-bp cDNA encoding the full-length MLK2 protein. The predicted MLK2 polypeptide has 954 amino acids and contains a src homology 3 (SH3) domain, a kinase catalytic domain, a double leucine zipper and basic domain, and a large Cterminal domain. The 22-amino-acid N-terminal region has four glutamic acid residues immediately following the initiator methionine. Beginning at amino acid 23, the 55-amino-acid SH3 domain contains a 5-amino-acid insert in a position corresponding to inserts of 6 and 15 residues in the SH3 domains of n-src and the phosphatidylinositol -7'-kinase. Adjacent to the SH3 domain is a kjnase catalytic domain with conserved motifs associated with both serinehhreonine and tyrosine specificity. Beginning nine residues C-terminal to the catalytic domain, there are two Icucine/isoleucine zippers separated by a 13-aminoacid spacer sequence and followed by a stretch of hasic residues. Thc polybasic sequence contains a motif that is similar to nuclear localisation signals from several proteins. The C-terminal domain is composed of 491 amino acids of which 17% are serine or threonine and l h % are proline. This domain also has a biascd ratio of basic to acidic amino acids with ii calculated pl of 9.38. When used as a probe to examine mRNA expression in human tissues, ii MLK2 cDNA hybridised to a species of 3.8 kb that was expressed at highest levels in RNA from brain and skeletal muscle. The 3454-bp cDNA was also used for fluorescence in situ hybridisation to localise the MLR2 gene to human chromosome 19 q13.2.

From receptors to stress-activated MAP kinases

“…In contrast to MAPKs and MAPKKs, the MAPKKKs in the JNK and p38 modules are highly divergent in structure and gene number. To date, eleven di erent MAPKKKs have been identi®ed as upstream activators of JNK pathway (Widmann et al, 1999); MEKK1 (Lange et al, 1993), MEKK2 (Blank et al, 1996), MEKK3 (Blank et al, 1996), MTK1/ MEKK4 (Takekawa et al, 1997;Gerwins et al, 1997), Tpl-2/Cot (Aoki et al, 1991;Salmeron et al, 1996), MUK/DLK/ZPK Holzman et al, 1994;Reddy and Pleasure, 1994), MLK-2/MST (Dorow et al, 1995;Hirai et al, 1997), MLK-3/SPRK/ PTK-1 (Rana et al, 1996;Gallo et al, 1994;Ing et al, 1994), TAK1 (Yamaguchi et al, 1995), ASK1/ MAPKKK5 Ichijo et al, 1997) and ASK2 (Saitoh and Ichijo, unpublished observation)/MAPKKK6 (Wang et al, 1998) have been shown to activate JNKs by overexpression (Figure 1). Of these, MEKK1, MEKK2, MEKK3 and Tpl-2 can also activate the ERK pathway, while only TAK1, ASK1 and MTK1 have been shown to strongly activate p38s as well.…”

MST/MLK2, a Member of the Mixed Lineage Kinase Family, Directly Phosphorylates and Activates SEK1, an Activator of c-Jun N-terminal Kinase/Stress-activated Protein Kinase

“…Furthermore, it may also depend on which MAPKKKs and MAPKKs are expressed in the cell. Both MST/MLK2 and SEK1/MKK4/JNKK are highly expressed in skeletal muscle and brain among the human tissues tested, implying that they play an important role in the activation of JNK/SAPK in these tissues (20,21). In addition, several tumor cell lines show high levels of MST expression, implying some function of MST/MLK2 in cellular transformation (20).…”

“…In addition to a unique kinase domain, MUK/ DLK/ZPK contains two leucine zipper-like structures located proximal to the C-terminal end of the catalytic domain. These features are common to members of the mixed lineage kinase (MLK) family, including MLK1 (19), MST/MLK2 (20,21), and SPRK/PTK1/MLK3 (22)(23)(24). Therefore, MUK/DLK/ZPK has been identified as the fourth member of the MLK family (16 -18).…”

Activation of the JNK pathway during dorsal closure in Drosophila requires the mixed lineage kinase, slipper

“…Slpr is most related to the human MLK family of three proteins with up to 56% amino acid identity within the N-terminal region, consisting of ∼510 amino acids including the kinase domain (Fig. 4) (Dorow et al 1993(Dorow et al , 1995Gallo et al 1994;Ing et al 1994;Katoh et al 1995). The structural features of MLK proteins have provided clues to their function and support their role as important signal transducers for the JNK pathway, conveying signals from upstream GTPases to downstream JNKKs, (Rana et al 1996;Teramoto et al 1996;Tibbles et al 1996;Hirai et al 1997;Merritt et al 1999).…”