A novel predicted calcium-regulated kinase family implicated in neurological disorders

Abstract

The catalogues of protein kinases, the essential effectors of cellular signaling, have been charted in Metazoan genomes for a decade now. Yet, surprisingly, using bioinformatics tools, we predicted protein kinase structure for proteins coded by five related human genes and their Metazoan homologues, the FAM69 family. Analysis of three-dimensional structure models and conservation of the classic catalytic motifs of protein kinases present in four out of five human FAM69 proteins suggests they might have retained catalytic phosphotransferase activity. An EF-hand Ca(2+)-binding domain in FAM69A and FAM69B proteins, inserted within the structure of the kinase domain, suggests they may function as Ca(2+)-dependent kinases. The FAM69 genes, FAM69A, FAM69B, FAM69C, C3ORF58 (DIA1) and CXORF36 (DIA1R), are by large uncharacterised molecularly, yet linked to several neurological disorders in genetics studies. The C3ORF58 gene is found deleted in autism, and resides in the Golgi. Unusually high cysteine content and presence of signal peptides in some of the family members suggest that FAM69 proteins may be involved in phosphorylation of proteins in the secretory pathway and/or of extracellular proteins.

Figure 1. CLANS graphs visualizing sequence similarities between protein kinase-like families.

Nodes represent sequences, edges represent similarity relationships. PSI-BLAST-detected significant (dark grey) and sub-significant (light grey) similarities shown. Dark green: pkinase and pkinase_Tyr families, Red: FAM69; dark blue: SELO; yellow: alpha kinase; brown: UL97; cyan: PPDK, magenta: PI3_PI4; light green: PIP5K, orange: DUF1193 (FAM20); Black: other kinase families. The following P-value thresholds for significance of sequence similarity were used: 0.1 (top left), 0.001 (top right), 1E-5 (bottom left) and 1E-10 (bottom right).

Figure 2. Multiple sequence alignments of selected FAM69 proteins.

Left: alignment of the kinase domain, covering the region 83–422 of FAM69A. The sequences are aligned using Promals3D (see Methods). Secondary structure prediction for human shown for selected proteins, for the solved structures actual secondary structure shown. Secondary structure elements named as in PKA . Locations of predicted key catalytic residues shown, in standard PKA numbering (e.g. D166), as well as the ATP-binding loop (GxGxxG). SwissProt identifiers shown for human sequences, otherwise, NCBI GI identifiers shown together with abbreviations of species names: Sp: sea urchin Strongylocentrotus purpuratus, Nv: sea cucomber Nematostella vectens, Dm: fruit fly Drosophila melanogaster; Mm: Mus musculus; Ce: Caenorhabditis elegans, Co: Capsaspora owczarzaki; S sp: Salpingoeca sp. ATCC 50818. Also shown selected close kinase homologues (PKDCC and SGK196) as well as sequences of selected kinases of known structures. Numbers in brackets indicate numbers of residues omitted from the alignment (shown only for the 1 cdk, 3 sxs structures, and for human FAM69A, DIA1, PKDCC and SGK196 sequences). R and C characters on black background above the alignment indicate the regulatory and catalytic spine residues, respectively . The location of the the EF-hand motif shown, the motif itself is excised from the alignment and shown on the right. Right: alignment of the EF-hand region (corresponding to the region 165–199 of human FAM69A). Also shown EF-hand regions of human calmodulin and the 2PMY region used for model building.

Figure 3. Kinase motifs.

Sequence logos for selected kinase motifs in the FAM69 family. Top: the β-4 - β-5 region. Middle row, left: the predicted active site region (corresponding to D166 and N171 in PKA). Middle row, right- the Mg²⁺ - binding motif (corresponding to the DFG motif in PKA). Bottom, left: predicted helix α-F. Bottom, right: predicted helix α-G.

Figure 4. Cysteines in kinases.

Histogram of cysteine residue count in kinase domains. Left scale, magenta bars: 516 kinase domains of the human kinome , Right scale, red bars: human FAM69 kinase domains; right scale, yellow bars: human FAM20 kinase domains.

Figure 5. Structure model of the kinase domain of human FAM69A.

Top left: model coloured by MetaMQAP model quality score (blue: good quality, red: poor quality). On left, the EF-hand motif is shown in yellow. Top right: as in Fig. 5 (top left), model coloured by sequence: from dark blue (N-terminus) to dark red (C-terminus). Bottom: close-up of the predicted active site with ATP molecule bound. Side chains of key predicted active site residues shown: D294 (PKA numbering: 166), N299 (171), D312 (184), also the two cysteines near the predicted active site that may form a S-S bridge: C293 and C331.