A Single Pair of Acidic Residues in the Kinase Major Groove Mediates Strong Substrate Preference for P-2 or P-5 Arginine in the AGC, CAMK, and STE Kinase Families

Abstract

Most basophilic serine/threonine kinases preferentially phosphorylate substrates with Arg at P؊3 but vary greatly in additional strong preference for Arg at P؊2 or P؊5. The structural basis for P؊2 or P؊5 preference is known for two AGC kinases (family of protein kinases A, G, and C) in which it is mediated by a single pair of acidic residues (PEN؉1 and YEM؉1). We sought a general understanding of P؊2 and P؊5 Arg preference. The strength of Arg preference at each position was assessed in 15 kinases using a new degenerate peptide library approach. Strong P؊2 or P؊5 Arg preference occurred not only in AGC kinases (7 of 8 studied) but also in calmodulin-dependent protein kinase (CAMK, 1 of 3) and Ste20 (STE) kinases (2 of 4). Analysis of sequence conservation demonstrated almost perfect correlation between (a) strong P؊2 or P؊5 Arg preference and (b) acidic residues at both PEN؉1 and YEM؉1. Mutation of two kinases (PKC-and p21-activated kinase 1 (PAK1)) confirmed critical roles of both PEN؉1 and YEM؉1 residues in determining strong R-2 Arg preference. PAK kinases were unique in having exceptionally strong Arg preference at P؊2 but lacking strong Arg preference at P؊3. Preference for Arg at P؊2 was so critical to PAK recognition that PAK1 activity was virtually eliminated by mutating the PEN؉1 or YEM؉1 residues. The fact that this specific pair of acidic residues has been repeatedly and exclusively used by evolution for conferring strong Arg preference at two different substrate positions in three different kinase families implies it is uniquely well suited to mediate sufficiently good substrate binding without unduly restricting product release.

SLK-mediated phosphorylation of paxillin is required for focal adhesion turnover and cell migration

“…The resulting reduction in acidic content negates the need for basic residues such as arginine, at P(-2) and P(-5) in the substrate. This however, is a requirement of the basophilic PAK subfamily [162,163]. Although some GCKs such as Mst1, still phosphorylate substrates containing a basic residue at P(-2), phosphorylation of the GCKs SPAK (Ste20/SPS1-related proline alanine-rich kinase) and OSR1 (oxidative stressresponse 1), further confirms that GCKs do not require basic residues in the consensus sequence of their substrates [161,162,164].…”

“…Since SLK is classified as a GCK-V kinase, our analysis of current Ste20 phosphorylation literature will focus on the GCK subfamily. An interesting trait of the GCK subfamily is the absence of an acidic residue within the catalytic loop of the kinase domain present within PAKs as an aspartic acid [162,163]. The resulting reduction in acidic content negates the need for basic residues such as arginine, at P(-2) and P(-5) in the substrate.…”

“…However, studies would suggest that GCKs are more highly regulated by the molecular interactions that increase substrate concentration than the requirement of a consensus sequence that may not apply to all substrates [49,163]. Interestingly, Pike et al (2008) shows SLK to require additional mechanisms in order to maintain itself in a dimerized and active state since activation segment phosphorylation appears to weaken SLK self-association [77].…”

Myosin 3A Kinase Activity Is Regulated by Phosphorylation of the Kinase Domain Activation Loop

“…The Thr-184 site is highly conserved in kinases that are activated by phosphorylation of the activation loop, including the well characterized STE20 member, PAK1. The Thr-184 site contains P(Ϫ2), P(Ϫ3), and P(Ϫ5) arginines (where P is associated with the residue position relative to the phosphorylation site at P0) that are associated with the known substrate specificity for other basophilic kinases such as PKA (37). Therefore, the Thr-184 site can likely be phosphorylated by other kinases as well as through autophosphorylation.…”

LOK is a major ERM kinase in resting lymphocytes and regulates cytoskeletal rearrangement through ERM phosphorylation

“…Most of the work in mammalian cells relates to candidates belonging to the AGC family (e.g., PKC and ROCK) but 2 considerations prompt looking beyond the AGC family for mammalian ERM kinases. First, typical kinases of the AGC family are basophilic kinases that virtually require an arginine residue in at least one critical position before the phosphorylated residue (especially P-3, but also P-2) and benefit from additional positively charged residues nearby (16,17). In contrast, the ERM phosphorylation site lacks R at either of these positions and has an unusual bulky aromatic residue at P-2.…”