MARK Interacts with MIK a GCKlike Kinase

While the mechanisms by which the animal atypical receptor kinases transduce signals are not completely elucidated, it has been proposed that the impaired kinase domains may interact specifically with other proteins upon ligand binding by their receptor domain (Stein and Staros, 2000). We have thus searched for proteins that could interact with MARK-KD by performing a two-hybrid screen of a 7 DAP maize embryo cDNA library cloned in the pACT2 vector with MARK-KD fused to the Gal4 DNA binding domain.

A partial cDNA clone, DH5, presenting an ORF with high sequence similarities to the C-terminal domain of Brassica and Arabidopsis MAP4K (Leprince et al., 1999) was isolated. The interaction between DH5 and MARK-KD was further analysed by pull-down experiments. Figure 7.4A shows that all the different deletions of the DH5 clone tested abolished the interaction. We next tested the role that the atypical residues in MARK-KD might have in the interaction with MIK. The substitution of the atypical amino-acids present in MARK-KD subdomains VIb and VII by those present in typical kinases does not significantly affect the interaction with MIK, suggesting that they do not play a major role in the interaction (Fig. 7.4B).

Fig. 7.3. Scheme representing the MARK protein. The relative position of the leucine-rich repeats (LRR), the transmembrane domain (TM) and the kinase-like domain (KD) are indicated, as are the positions of the 11 subdomains conserved among kinases. The sequences of the subdomains III, VIb and VII of MARK-KD are shown compared with those of CLV1 and SRK receptor kinases, and the amino acids that are invariable among kinases are highlighted.

Fig. 7.3. Scheme representing the MARK protein. The relative position of the leucine-rich repeats (LRR), the transmembrane domain (TM) and the kinase-like domain (KD) are indicated, as are the positions of the 11 subdomains conserved among kinases. The sequences of the subdomains III, VIb and VII of MARK-KD are shown compared with those of CLV1 and SRK receptor kinases, and the amino acids that are invariable among kinases are highlighted.

Fig. 7.4. GST pull-down experiments performed between MARK-KD and MIK. (A) The complete DH5-MIK sequence, as well as different deletions of DH5-MIK (a-c), were fused to GST and immobilized on to glutathion-S-Sepharose and were incubated with recombinant His-tagged MARK-KD, using GST as a negative control, in binding buffer (Hepes pH 7.9 20 mM, glycerol 20%, EDTA 0.2 mM, NaCl 150 mM, NP40 0.5%, PMSF 0.1 mM, DTT 0.5 mM). After extensive washing with the same buffer, proteins were eluted with Laemmli buffer, separated by SDS-PAGE and transferred to nitrocellulose. Immunodetection was performed with an anti-MARK polyclonal antibody. (B) Immobilized GST and GST fused MARK-KD, as well as three different MARK-KD mutants (A-C) were incubated with His-tagged DH5-MIK as described in (A). Immunodetection was performed with an anti-DH5-MIK polyclonal antibody.

Fig. 7.4. GST pull-down experiments performed between MARK-KD and MIK. (A) The complete DH5-MIK sequence, as well as different deletions of DH5-MIK (a-c), were fused to GST and immobilized on to glutathion-S-Sepharose and were incubated with recombinant His-tagged MARK-KD, using GST as a negative control, in binding buffer (Hepes pH 7.9 20 mM, glycerol 20%, EDTA 0.2 mM, NaCl 150 mM, NP40 0.5%, PMSF 0.1 mM, DTT 0.5 mM). After extensive washing with the same buffer, proteins were eluted with Laemmli buffer, separated by SDS-PAGE and transferred to nitrocellulose. Immunodetection was performed with an anti-MARK polyclonal antibody. (B) Immobilized GST and GST fused MARK-KD, as well as three different MARK-KD mutants (A-C) were incubated with His-tagged DH5-MIK as described in (A). Immunodetection was performed with an anti-DH5-MIK polyclonal antibody.

The DH5 clone was used as a probe to screen maize genomic and cDNA libraries and the corresponding full-length and genomic clones were obtained. The deduced protein sequence, MIK, shows high similarities with the GCK subfamily of the Ste20 family of MAPkinases. Some of the Ste20-related proteins have been shown to phosphorylate MAPKKK, thus acting as MAP4K (Drogen et al., 2000; Raitt et al, 2000). The GCK subfamily consists of a heterogeneous group of proteins that, in most cases, are still poorly characterized. Nevertheless, some GCKs have been shown to connect cell-surface receptors to intracellular MAPK cascades (Kyriakis, 1999), suggesting that MIK could connect MARK to intracellular signalling cascades.

The expression pattern of MIK is consistent with a possible interaction with MARK, as both have a similar pattern of expression during embryogenesis, presenting a peak of expression at 15 DAP in embryo and endosperm (data not shown). Moreover, immunolocalizations of the MIK protein have shown that it accumulates preferentially in provascular cells of the coleoptile and radicle - cells that also exhibit a high accumulation of the MARK protein.

GCK proteins contain an amino-terminal kinase domain and a carboxyl-terminal regulatory domain that probably inhibits its kinase activity. The interaction shown here of the intracellular inactive kinase domain of MARK with the C-terminal regulatory domain of MIK (MIK-RD) could thus suggest a model in which, upon ligand binding, MARK-KD would interact with

MIK-RD, releasing the inhibition of the kinase activity of MIK and allowing this protein to activate intracellular signalling cascades.

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