Comprehensive profiling of the STE20 kinase family defines features essential for selective substrate targeting and signaling output

Abstract

Specificity within protein kinase signaling cascades is determined by direct and indirect interactions between kinases and their substrates. While the impact of localization and recruitment on kinase–substrate targeting can be readily assessed, evaluating the relative importance of direct phosphorylation site interactions remains challenging. In this study, we examine the STE20 family of protein serine–threonine kinases to investigate basic mechanisms of substrate targeting. We used peptide arrays to define the phosphorylation site specificity for the majority of STE20 kinases and categorized them into four distinct groups. Using structure-guided mutagenesis, we identified key specificity-determining residues within the kinase catalytic cleft, including an unappreciated role for the kinase β3–αC loop region in controlling specificity. Exchanging key residues between the STE20 kinases p21-activated kinase 4 (PAK4) and Mammalian sterile 20 kinase 4 (MST4) largely interconverted their phosphorylation site preferences. In cells, a reprogrammed PAK4 mutant, engineered to recognize MST substrates, failed to phosphorylate PAK4 substrates or to mediate remodeling of the actin cytoskeleton. In contrast, this mutant could rescue signaling through the Hippo pathway in cells lacking multiple MST kinases. These observations formally demonstrate the importance of catalytic site specificity for directing protein kinase signal transduction pathways. Our findings further suggest that phosphorylation site specificity is both necessary and sufficient to mediate distinct signaling outputs of STE20 kinases and imply broad applicability to other kinase signaling systems.

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

“…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.…”

Mob Family Proteins: Regulatory Partners in Hippo and Hippo-Like Intracellular Signaling Pathways

“…Fly Hippo kinase is a member of the STE20 kinase family, which is grouped together based on sequence similarity in their kinase domain, and on conservation of an auto-phosphorylation consensus sequence that is essential for their activation (Ling et al, 2008;Record et al, 2010). The large STE20 family is divided into the P21-Activated Kinase (PAK) and the GCK sub-families (Dan et al, 2001;Record et al, 2010;Miller et al, 2019;reviewed in Thompson and Sahai, 2015). Only the GCK sub-family kinases have been implicated in Mob or NDR kinase function.…”

Identification of a dual TAOK1 and MAP4K5 inhibitor using a structure-based virtual screening approach

“…The STE20 (Sterile 20) protein kinase family consists of a total of 30 serine-threonine kinases that can be grouped into 10 subfamilies 12 . This kinase family has been implicated in the regulation of cytoskeletal rearrangements and actin stress fibre disassembly 13 and modulation of the MAPK cascade stress responses can have an impact on cellular processes, such as proliferation, cell cycle, apoptosis and differentiation 14,15 .…”