Analogous regulatory sites within the alphaC-beta4 loop regions of ZAP-70 tyrosine kinase and AGC kinases

Abstract

The precise positioning of the flexible C-helix in the catalytic core is a critical step in the activation of most protein kinases. Consequently, the alphaC-beta4 loop, which anchors the C-helix to the catalytic core, is highly conserved and mediates key structural interactions that serve as a hinge for C-helix movement. While these hinge interactions are conserved across diverse eukaryotic protein kinase structures, some families such as AGC kinases diverge from the canonical hinge interactions. This divergence was recently proposed to facilitate an alternative mode of regulation wherein a conserved C-terminal tail interacts with the alphaC-beta4 loop to position the C-helix. Here we show how interactions between the alphaC-beta4 loop and the N-terminal SH2 domain of ZAP-70 tyrosine kinase are mechanistically and functionally analogous to interactions between the alphaC-beta4 loop and the C-terminal tail of AGC kinases. Such cis regulation of protein kinase activity may be a feature of other eukaryotic protein kinase families as well.

Figure 1

Selective conservation of the HxN motif in the αC-β4 loop region and its structural location in Cdk2 (EPK) and aminoglycoside kinase (ELK). A. A contrast hierarchical alignment showing evolutionary constraints imposed within the C-helix and the αC-β4 region of EPK's. All EPK sequences (14,667) constitute the foreground set in the alignment, while all ELK sequences constitute the background set. Representative sequences from various EPK families are used as the display set. The histogram above the alignment measures the degree to which aligned residue positions in the foreground set are shifted away from the corresponding position in the background set. Residue positions subject to the strongest constraints are highlighted with chemically similar conserved amino acid residues colored similarly; very weakly conserved positions and nonconserved positions are shown in dark and light grey, respectively. Dots below the histograms indicate those residues positions that most strikingly distinguish EPKs from ELKs, as selected by our statistical procedure[9]. The foreground set includes both EPK sequences shown in the alignment and other EPK sequences, the numbers of which (after adjusting for sequence redundancy) are denoted by their weighted residue frequencies (‘wt_res_freqs’) below the alignment. Residue frequencies are indicated in integer tenths where, for example, a ‘7’ indicates that the corresponding residue directly above it occurs in 70-80% of the (weighted) sequences. Similarly, the background set (ELKs) is shown below the foreground alignment. NCBI sequence identifiers used in alignment 1A are: 6730497|Cdk2-human; 20150484|Cdk6-human; 1110512|Erk2-rat; 24987248|Gsk3b-human; 6137569|FGF-human; 15988251|Tie2-human; 30749934|C-Abl-mouse; 3114436|CalMK-rat; 46397802|Aurora1K-yeast; 38016021|STK-plant; 47169341|B-Raf-human; 15988011|TGF-human. B. Structural interactions that anchor the αC-β4 loop to the C-lobe in the active state of Cdk2 (PDB: 1QMZ). C. Structural interactions in the αC-β4 region of inactive Cdk2 (PDB: 1HCL). E. The αC-β4 loop associated interactions in aminoglycoside kinase (PDB: 1J7L). The structural figures shown in Fig 1(B-E) and Fig 2(C-D) were generated using Pymol [20]. Hydrogen bonds are indicated by dotted lines and residues are colored using a coloring scheme as indicated in the alignments. The EPK-ELK shared residues are shown in magenta. EPK-specific residues are shown in gold. Group specific residues are shown in yellow, while family specific residues are shown in cyan. The C-helix is denoted as αC.

Figure 2

Sequence variation within the αC-β4 loop region reflects functional variation in AGC kinases and ZAP-70 tyrosine kinase A. Contrast hierarchical alignment showing evolutionary constraints imposed within the αC-β4 region of AGC kinases. These constraints were measured using all AGC kinase sequences as the foreground set and all eukaryotic protein kinase sequences as the background set. Representative sequences from various AGC kinase families constitute the display set. NCBI sequence identifiers used in alignment 2A are: 125205|PKA_human; 1170703|PKB_human; 20141582|PKCT_human; 417080|GRK_fruitfly; 47605999|RHOK_human; 7649389|RSK_plant; 56749457|NDRK-human B. Evolutionary constraints imposed within the αC-β4 region of ZAP70 kinases and related Syk kinases. In this alignment, the foreground set corresponds to ZAP-70 and Syk tyrosine kinase sequences, while the background corresponds to all tyrosine kinases. Representative sequences from different phyla of ZAP-70 and Syk constitute the display set. NCBI sequence identifiers used in alignment 2B are: 1177044|ZAP70-human; 26453338|ZAP70-mouse; 50416266|ZAP70-frog; 50761018|ZAP70-chicken; 47086347|Syk-zebrafish; 50761846|Syk-chicken C. Backbone and side-chain interactions in the αC-β4 region of PKA showing the integration of the C-terminal tail with the hinge region. The hydrophobic motif (HF-motif) in the C-tail and its docking interaction with the C-helix is also shown (PDB: 1ATP). D. Specific interactions between the SH2 domain and the αC-β4 region of ZAP70 tyrosine kinase (PDB: 2OZO). Residue coloring are as indicated in Fig 1 caption.