Expanding the Mitogen-Activated Protein Kinase (MAPK) Universe: An Update on MAP4Ks

Phosphorylation-mediated signaling cascades control plant growth and development or the response to stress conditions. One of the best studied signaling cascades is the one regulated by MITOGEN-ACTIVATED PROTEIN KINASEs (MAPKs). However, MITOGEN-ACTIVATED PROTEIN KINASE KINASE KINASE KINASEs (MAP4Ks) are hardly explored. Here, we will give a comprehensive overview of what is known about plant MAP4Ks and highlight some outstanding questions associated with this largely uncharacterized class of kinases in plants.


INTRODUCTION
To regulate their growth and development or respond to stress conditions, plants use-like many other organisms-phosphorylation-mediated signaling cascades. The central enzymes in these cascades are kinases, which-via reversible phosphorylation-mediate protein folding (conformation), protein function and the regulation of enzymatic activities, define substrate specificity, and influence protein localization, complex formation, and degradation (Stone and Walker, 1995). The Arabidopsis genome contains more than 1,000 kinases (Wang et al., 2003;Wang et al., 2007;Dissmeyer and Schnittger, 2011), including a largely uncharacterized class of mitogenactivated protein kinase kinase kinase kinases (MAP4Ks) (Champion et al., 2004). Here, we will give a comprehensive overview of what is known about plant MAP4Ks and highlight some outstanding questions.

THE CANONICAL MAPK SIGNALING MODULE
One of the best studied signaling cascades is the one regulated by MITOGEN-ACTIVATED PROTEIN KINASEs (MAPKs). The MAPK cascade is conserved in yeast, insects, nematodes, plants, and mammals, and its main role is to modulate protein function through linear sequential serine/threonine and/or tyrosine phosphorylation (Leprince et al., 1999;Dan et al., 2001;Champion et al., 2004;Cakir and Kilickaya, 2015). The canonical MAPK signaling module is composed of a MAPK KINASE KINASE (MAP3K), a MAPK KINASE (MAP2K), and a MAPK (Dan et al., 2001;Colcombet and Hirt, 2008) (Figure 1A). In a typical MAPK cascade, a MAP3K specifically activates a dual specific MAP2K by phosphorylation, which in turn activates a MAPK by phosphorylation of threonine and tyrosine residues Krysan and Colcombet, 2018). Given the number of MAP3Ks, MAP2Ks, and MAPKs in Arabidopsis thaliana (Jonak, 2002;Champion et al., 2004;Colcombet and Hirt, 2008;Krysan and Colcombet, 2018), the theoretical MAPK network contains an enormous number of possible combinations.

A NEW HOPE: THE MAP4K FAMILY IN PLANTS
However, in contrast to the activation mechanisms described above, a MAP3K is sometimes phosphorylated by a MAP4K (Dan et al., 2001;Champion et al., 2004) ( Figure 1A). For example, yeast Ste20 acts as a MAP4K that directly phosphorylates Ste11, a MAP3K in the mating pathway (Wu et al., 1995), the mammalian Hematopoietic progenitor kinase (HPK1) phosphorylates the MAP3K MEKK1 (Dan et al., 2001;Chuang et al., 2016), and PAK2 activates the MAP3K Raf-1 by direct phosphorylation (Dan et al., 2001;Chuang et al., 2016). The yeast Ste20-related MAP4K family is evolutionarily conserved (Wu et al., 1995;Champion et al., 2004). In non-plant organisms, the MAP4K family is divided in two families according to the location of the kinase domain: (i) p21-activated kinases (PAKs), with a C-terminal kinase domain and an N-terminal GTPase-binding domain, and (ii) germinal center kinase (GCKs), with an N-terminal kinase domain and lacks the GTPase-binding domain (Dan et al., 2001).
In Brassica napus, BnMAP4Ka1 and BnMAP4Ka2 were isolated from a globular/heart stage embryo cDNA library (Leprince et al., 1999). These two putative proteins contain 12 serine/threonine protein kinase catalytic subdomains at the Nterminus, possess long disordered sequences at the C-terminus, and are similar to the GCK subfamily of yeast Ste20-like MAP4Ks (Leprince et al., 1999) (Figure 1B). BnMAP4Ka1 and BnMAP4Ka2 are mainly expressed in flower buds, siliques, different stages of embryogenesis, and roots (Leprince et al., 1999).

MAIZE ATYPICAL RECEPTOR KINASE (MARK)-INTERACTING KINASE (MIK), a GCK-III subfamily MAP4K
from Zea mays, possesses an N-terminal kinase domain that displays high similarity to the GCK subfamily of yeast Ste20-like MAP4Ks (Llompart et al., 2003) ( Figure 1B). Moreover, the kinase domain of MIK contains a VGTPFWMAPEV sequence, which aligns with the signature motif of Ste20-like kinases (Dan et al., 2001;Llompart et al., 2003), and both the N-terminal kinase domain and the C-terminal part share high sequence similarity with AtMAP4Ka1, AtMAP4Ka2, BnMAP4ka1 and BnMAP4ka2 (Llompart et al., 2003). MIK interacts with MARK, which is an atypical receptor kinase expressed during embryogenesis and in the meristems of adult maize plants, and this interaction increases the activity of MIK (Llompart et al., 2003;Castells et al., 2006) (Figure 1A). Different isoforms of MIK show variable kinase activity and are differentially activated through the interaction with MARK, suggesting that the kinase activity of MIK is also regulated by alternative splicing (Castells et al., 2006).
In Solanum chacoense, ScMAP4K1 is expressed during fertilization and early embryogenesis (Major et al., 2009). Phylogenetic analysis revealed that ScMAP4K1 belongs to the GCK-II subfamily and is the ortholog of Arabidopsis SIK1/ MAP4K3 (Major et al., 2009). However, unlike the GCK-II members in mammals, ScMAPK1 has a central kinase domain (Major et al., 2009). While ScMAP4K1 is strongly expressed in reproductive tissues (such as pollen and pollen tubes) (Wu et al., 1995), full length ScMAP4K1 is not present in both pollen and pollen tubes, suggesting that ScMAP4K1 is also regulated by alternative splicing, similar to MIK in maize (Castells et al., 2006;Major et al., 2009). Interestingly, ScMAP4K1 RNAi lines show altered ovule, seed and fruit development, indicating that ScMAP4K1 plays a vital role in those processes (Major et al., 2009).

THE ARABIDOPSIS MAP4KS AWAKEN
There are 10 MAP4Ks in Arabidopsis (Champion et al., 2004), but while plant MAP4Ks have been described already at the end of the 20 th century, it is only since 2013 that functional characterization in A. thaliana has begun (Takemiya et al., 2013a, Xiong et al., 2016. Most of the Arabidopsis MAP4Ks have an N-terminal catalytic kinase domain, but SERINE/ THREONINE KINASE 1 (SIK1)/MAP4K3 has a more centrally located kinase domain (Xiong et al., 2016;Zhang et al., 2018) (Figure 1B). Phylogenetic analysis based on the MAP4K kinase domain showed that MAP4Ka1 and MAP4Ka2 are GCK-III subfamily members, that SIK1 belongs to the GCK-II subfamily, and that other Arabidopsis MAP4Ks are part of the GCK-VI subfamily (Major et al., 2009) (Figure 1B).
Arabidopsis SERINE/THREONINE KINASE 1 (SIK1)/ MAP4K3 encodes a MAP4K kinase that contains a central kinase domain and that is highly conserved in land plants ( Figure 1B) (Zhang et al., 2018). SIK1 complements the ste20D yeast mutant phenotypes with respect to bud site selection and mitotic exit (Xiong et al., 2016), confirming that SIK1 is similar to Ste20 and belongs to the MAP4K family (Xiong et al., 2016).
SIK1 is strongly expressed in mature organs and tissues, such as developed vascular tissues, stipules of true leaves, mature trichrome, and guard cells (Xiong et al., 2016). The sik1 mutants are dwarfed and grow slow compared to wild type and display a shorter primary root and shorter root hairs, a reduced rosette leaf area, reduced area and number of the petal cells of fully opened flower, and reduced plant height, smaller siliques, and smaller seeds (Xiong et al., 2016). The abovementioned sik1 mutant phenotypes are caused by a reduced cell number and size, indicating that SIK1 influences growth via regulating cell proliferation and cell expansion (Xiong et al., 2016). In agreement with this, the expression of SIK1 orthologs BnMAP4Ka1 and BnMAP4Ka2 is cell cycle-dependent and follows the same expression pattern as a G2-phase marker (Leprince et al., 1999). Thus, resembling the function of Ste20, SIK1 plays a role as a positive regulator of cell cycle exit (Xiong et al., 2016).
Interestingly, the N-terminal part of SIK1 interacts with MOB KINASE ACTIVATOR 1A (MOB1A) and MOB1B (Xiong et al., 2016). In mammals, Mob1 is involved in cell proliferation and tumor suppression and controls appropriate cell numbers and organ size (Pan, 2010). Also in Arabidopsis, MOB1A regulates organ growth, root tip tissue patterning, cell number and size, and sporophyte and gametophyte development (Galla et al., 2011;Pinosa et al., 2013). SIK1 is localized at the plasma  membrane and in the trans-Golgi network/early endosome (TGN/EE), and the interaction of SIK1 and MOBs results in translocation of SIK1 to the nucleus (Xiong et al., 2016). In humans, MAP4Ks are involved in immunity and activate nuclear factor kB immune signaling (Chuang et al., 2016). Indeed, also in Arabidopsis, MAP4Ks such as SIK1 and MAP4K4 function in immunity signaling (Zhang et al., 2018;Jiang et al., 2019) ( Figures 1A and 2B). Both SIK1 and MAP4K4 directly interact with, phosphorylate, and stabilize the immunity regulator BOTRYTIS-INDUCED KINASE 1 (BIK1) (Zhang et al., 2018;Jiang et al., 2019). Moreover, SIK1 directly interacts with and phosphorylates RBOHD upon flg22 perception (Zhang et al., 2018). In addition, PP2C38, a protein phosphatase, dephosphorylates BIK1 and maintains a minimum BIK1 phosphorylation status in the absence of flg22 (Couto et al., 2016). MAP4K4, which is localized in the cytosol and at the plasma membrane, phosphorylates PP2C38 in the presence of flg22, and phosphorylated PP2C38 disassociates from BIK1, indicating that MAP4K4 also regulates BIK1 activity through controlling PP2C38 (Jiang et al., 2019).

THE RISE OF MAP4K FEATURES
Plant MAP4Ks obviously contain several kinase-specific features, such as (i) an invariant lysine residue, an HRD motif, and a DFG motif, which contribute to ATP binding and regulate the catalytic activity of the protein kinase (Carrera et al., 1993;Jiang et al., 2019;Takemiya et al., 2013a;Xiong et al., 2016;Zhang et al., 2018), (ii) a glycine-rich loop with a GXGXXG motif, a structural hallmark of protein kinases (Zhang et al., 2018) (Figure 2C). In addition, the GTPyWMAPEv motif (a small letter indicates less conservation) in subdomain VIII of the kinase domain, termed the Ste20 signature sequence, is the primary reason to classify them as MAP4Ks (Dan et al., 2001;Jonak, 2002;Llompart et al., 2003) ( Figure 2C). Furthermore, most plant MAP4Ks possess a long C-terminal region that is largely disordered (Champion et al., 2004;Major et al., 2009) ( Figure 2C). Ste20 kinases also often contain proline-rich motifs (Leprince et al., 1999;Chuang et al., 2016;Miller et al., 2019), and these were also described for BnMAP4K1 and BnMAP4K2 (Leprince et al., 1999). While some non-plant MAP4Ks contain a C-terminal citron-homology domain (Chuang et al., 2016), this was not reported for plant MAP4Ks.

CONCLUSION AND PERSPECTIVES
While our knowledge on plant MAP4Ks is gradually increasing, there is still very little known about them ( Figure 1A). One of the main outstanding questions-also for non-plant MAP4Ks-is what their substrates are and if they thus can all be considered as true MAP4Ks. More and more evidence is accumulating that these MAP4Ks also have other targets than MAP3Ks (Plouffe et al., 2016;Xiong et al., 2016;Bae and Luo, 2018;Zhang et al., 2018;Jiang et al., 2019). And, there is-so far-no evidence that plant MAP4Ks act on MAP3Ks. Taken together, this might warrant revisiting the MAP4K name. Second, we lack a comprehensive overview of the MAP4Ks in the green lineage, which would facilitate defining common characteristics. Third, using current methodologies to explore kinase signaling (Zhang et al., 2016) is an essential next step to deepen our insight not only regarding the substrates, but also with respect to potential protein complexes that are formed. Fourth, the subcellular localization of MAP4Ks spans the plasma membrane, cytoplasm, and nucleus, but the precise role in these compartments has hardly been investigated.

AUTHOR CONTRIBUTIONS
LP and IS organized and wrote the manuscript.

FUNDING
LP was supported by a grant from the Chinese Scholarship Council.