Recognition of physiological phosphorylation sites by p21-activated kinase 4
Graphical abstract
Introduction
For protein kinases to correctly propagate signals, they must selectively target their downstream substrates. This can occur through an array of mechanisms including co-localization, docking interactions (Ubersax and Ferrell, 2007), and direct binding of the kinase catalytic domain to the substrate (Miller and Turk, 2018). Among these interactions, short linear motifs comprising amino acids that flank the substrate residue can significantly contribute to selectivity for both serine/threonine and tyrosine kinases (Miller and Turk, 2018, Turk, 2008). The phosphoacceptor residue itself is also a key component of substrate specificity; for example, tyrosine kinases are structurally distinct from serine/threonine kinases (Taylor et al., 1995, Ubersax and Ferrell, 2007). Similarly, serine/threonine kinases often discriminate between serine and threonine residues, which is driven by the residue immediately following the conserved activation segment DFG motif, termed the DFG + 1 residue (Chen et al., 2014). While most protein kinases display strong phosphorylation site preferences in vitro, many bona fide protein substrates are phosphorylated at sites that would appear to be disfavored by the kinase (Chen et al., 2014, Pinna and Ruzzene, 1996). The mechanisms of this apparent disfavored phosphorylation are not generally well understood, so in this study we sought to understand an important disfavored kinase-substrate pair.
The p21-activated kinases (PAKs), are members of the sterile-20 family of serine-threonine kinases and are major downstream effectors of the Rho family of small GTPases (Arias-Romero and Chernoff, 2008, Ha et al., 2015, Hofmann et al., 2004, King et al., 2014, Kumar et al., 2017, Wells and Jones, 2010). The six members of the PAK group are classified by their domain organization into two sub-groups, the type I (PAK1-3) and type II (PAK4-6) PAKs, all of which play roles in cytoskeletal remodeling, cell motility, inhibition of apoptosis and transcription regulation (Ha et al., 2015, Kumar et al., 2017, Li et al., 2012). Of the type II PAKs, PAK4 is the best studied and has demonstrated roles in embryonic and neural development (Dart and Wells, 2013, Wells et al., 2010); its overexpression is also linked to tumorigenesis and metastasis, particularly in prostate and non-small cell lung cancers (Ahmed et al., 2008, Cai et al., 2015, Ha et al., 2015, Lu et al., 2017, Minden, 2012, Thillai et al., 2018, Wells et al., 2010).
All six PAKs strongly favor phosphorylation of serine over threonine, with type II PAKs including PAK4 appearing more selective than type I PAKs (Rennefahrt et al., 2007). Although PAK4 has multiple validated direct substrates (Table 1) (Bompard et al., 2010, Callow et al., 2002, Dan et al., 2001, Guo et al., 2014, Li et al., 2012, Li et al., 2010, Wells et al., 2010, Xu et al., 2016, Zhao et al., 2017, Zhuang et al., 2015), only one of these substrates is phosphorylated on a threonine residue, Thr508 of LIM domain kinase 1 (LIMK1) (Dan et al., 2001). This residue is phosphorylated by PAK4 (and other Rho-effector kinases) (Bernard, 2007, Scott and Olson, 2007), and its phosphorylation is required for full activation of LIMK1, which in turn phosphorylates and inactivates cofilin proteins - actin binding proteins that facilitate actin filament severing (Dan et al., 2001, Hamill et al., 2016). PAK4 therefore has an unusual exception to its strong preference for phosphorylation of serine substrates. The molecular mechanisms for this exception are unclear, especially because previous studies suggested the large and hydrophobic phenylalanine residue at PAK4′s DFG + 1 position should strongly disfavor phosphorylation of LIMK1-Thr508 (Chen et al., 2014).
To understand the mechanisms of PAK4′s atypical phosphorylation of LIMK1, we conducted a structural and functional study. We determined a 1.9 Å crystal structure of PAK4 catalytic domain in complex with a synthesized peptide corresponding to LIMK1′s substrate sequence (Thr508), and we compared this to a 2.2 Å crystal structure of PAK4 catalytic domain in complex with a synthesized peptide corresponding to a well characterized serine substrate of PAK4, β-catenin (Ser675). We find that similar to optimized ‘PAKtide’ substrates (Chen et al., 2014), the DFG + 1 residue is re-oriented to accommodate the unusual Thr phosphoacceptor. We then assessed the kinetics of kinase activity for PAK4 against LIMK and β-catenin substrate peptides with either Ser or Thr as the phosphoacceptor residue. We find that phosphoacceptor identity increases catalytic efficiency but does not affect Km, and although catalytic efficiency of wild-type LIMK1 and β-catenin are equivalent, T508S mutation of the LIMK1 peptide creates a highly efficient substrate.
Section snippets
Protein Expression and Purification
PAK4 expression and purification was conducted as previously described (Chen et al., 2014, Ha et al., 2012, Zhang et al., 2018). The catalytic domain of PAK4 (residues 109–426) (UniProt ID: O96013-2) was expressed using a modified pET28 vector with hexa-histidine (6xHis) tag, removable by TEV protease. Following nickel-affinity chromatography using a HisTrap chelating column (GE Healthcare) and gel filtration using a Superdex 200 10/300GL (GE Healthcare), purified PAK4 catalytic domain was
Results
To date, no structures have been determined of PAK family members in complex with regions of physiologically relevant substrates. Almost all type II PAK substrates are phosphorylated on a serine residue, with the sole exception of LIM domain kinase 1, which is phosphorylated on its activation loop threonine (Thr508) (Table 1, Fig. 1A). This exception to the strong preference of type II PAKs towards serine as a substrate caused us to wonder whether there is a structural basis for this
Discussion
PAK4 phosphorylates all of its known substrates on serine with the sole known exception of LIM domain kinase 1, which it phosphorylates on threonine. In this study we sought to understand the molecular and enzymatic reasons for these exceptions by studying PAK4 phosphorylation of a well-known serine substrate, β-catenin, and comparing this to its unusual substrate, LIMK1. We reveal the molecular basis for selectivity by showing that the DFG + 1 residue is important for discriminating between
Data availability
Coordinates and structure factors have been deposited in the Protein Data Bank under accession codes 6WLX and 6WLY. X-ray diffraction images are available online at SBGrid Data (Meyer et al., 2016): doi: https://doi.org//10.15785/SBGRID/781 (6WLX) and doi: https://doi.org//10.15785/SBGRID/782 (6WLY).
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
We thank Chad Miller for preparation of PAK4 protein used in this study. Staff at beamline 24-ID-E (NE-CAT-E) at the Advanced Photon Source, Argonne National Laboratory are thanked. This work is based upon research conducted at the Northeastern Collaborative Access Team beamlines, which are funded by National Institutes of Health grant P41GM103403. This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE
Declaration Competing Interests
The authors declare no competing financial interests.
Author contributions
Data acquisition: A.K.C.; Supervision of data acquisition: B.H.H., J.A.S.; Data analysis and interpretation: A.K.C., B.H.H., J.A.S., B.E.T., T.J.B.; Manuscript drafting: A.K.C., B.H.H., J.A.S., B.E.T., T.J.B.; Study conception and design: T.J.B.
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