Structural Basis for the Interaction between Pyk2-FAT Domain and Leupaxin LD Repeats

Proline-rich tyrosine kinase 2 (Pyk2) is a nonreceptor tyrosine kinase and belongs to the focal adhesion kinase (FAK) family. Like FAK, the C-terminal focal adhesion-targeting (FAT) domain of Pyk2 binds to paxillin, a scaffold protein in focal adhesions; however, the interaction between the FAT domain of Pyk2 and paxillin is dynamic and unstable. Leupaxin is another member in the paxillin family and was suggested to be the native binding partner of Pyk2; Pyk2 gene expression is strongly correlated with that of leupaxin in many tissues including primary breast cancer. Here, we report that leupaxin interacts with Pyk2-FAT. Leupaxin has four leucine–aspartate (LD) motifs. The first and third LD motifs of leupaxin preferably target the two LD-binding sites on the Pyk2-FAT domain, respectively. Moreover, the full-length leupaxin binds to Pyk2-FAT as a stable one-to-one complex. Together, we propose that there is an underlying selectivity between leupaxin and paxillin for Pyk2, which may influence the differing behavior of the two proteins at focal adhesion sites

Summary of SNVs in the first 2109 bp of the <i>GNAI3</i> non-coding exon 9/3’ UTR in patients affected with ocular albinism.

<p>Summary of SNVs in the first 2109 bp of the <i>GNAI3</i> non-coding exon 9/3’ UTR in patients affected with ocular albinism.</p

Summary of <i>GNAI3</i> gene exons, introns and 5’ and 3’ UTR sequences (ENST00000369851) in the 5' to 3' direction (Ensembl GRCh38/hg38).

<p>Summary of <i>GNAI3</i> gene exons, introns and 5’ and 3’ UTR sequences (ENST00000369851) in the 5' to 3' direction (Ensembl GRCh38/hg38).</p

<i>GNAI3</i> gene locus in human Chr1, Agilent Haloplex amplicons and Median coverage of <i>GNAI3</i>.

<p><b>A</b>.<i>GNAI3</i> is located in the p arm of chromosome 1, band 13.3 (red line). <b>B.</b> Agilent Haloplex Amplicons designed to amplify the <i>GNAI3</i> 9 exons, introns and 100 bp of each 5’ and 3’ UTRs are depicted in green and show that all regions of the gene were effectively covered. The <i>GNAI3</i> RefSeq is shown in black and was aligned against the GRCh37/hg19 human genome using the UCSC-BLAT browser. <b>C.</b> The median coverage level varied from 14 to 8,009 across the targeted <i>GNAI3</i> region analyzed in the HaloPlex design; only 1.2% of the target <i>GNAI3</i> region was not covered.</p

Validation of HaloPlex heterozygous insertion (c.*1994_*1995insA) of Patient 18 in the non-coding exon 9/3’ UTR of <i>GNAI3</i> by Sanger Sequencing.

<p>As in the previous figures, the track annotation panel depicts the Chr1 location of the insertion at position 109594317, followed by the forward sequences of the reference (R) and test sample (S), the confidence score peaks and reverse reference and test sample sequences. The red arrows point to the insertion in the sequences.</p

Validation of HaloPlex homozygous deletion (c.*1379_*1380delAT) of Patient 22 in the non-coding exon 9/3’ UTR of <i>GNAI3</i> by Sanger Sequencing.

<p>In addition to the track annotation panel clearly indicating the deletion at positions 109593703–109593704, the figure shows the forward sequences of the reference (R) and test sample (S) and the reverse sequences. The red arrows point to the deleted nucleotides.</p

Summary of variants in exons of <i>GNAI3</i> with likely functional effects in patients diagnosed with ocular albinism.

<p>Summary of variants in exons of <i>GNAI3</i> with likely functional effects in patients diagnosed with ocular albinism.</p

Structural representation of heterotrimeric GNAI3.

<p><b>A)</b> In the Gαβγ model, two domains of Gα are depicted: the Gα-helical insertion domain (Gα_AH) is in cyan and the Ras-like GTPase domain (Gα_Ras) is in gold; the Gβ subunit is in green, and the Gγ subunit in blue. Sites of observed amino-acid mutations are indicated with red spheres. B) Magnified representation of mutations D102E and V109F as well as of amino-acid R105. Wild-type side chain carbons are in cyan while mutated residues are in red. C) Magnified representation of the “T-shaped” π-stacking interaction between F223 and F250 in wild-type GNAI3 (gold) that is lacking in the F223V mutant (red). The distance from the <i>ortho</i> carbon on F250 to the centroid of F223 is 3.5 Å, shown with a dotted black line. D) Magnified representation depicting the “parallel displaced” π-stacking interaction between α-H213 (gold) and β-W332 (green) that is absent in the H213L mutant (red). The distance between the centroids of the aromatic rings is 4.8 Å, shown with a dotted black line.</p

Primers used in Sanger sequencing for validation of identified HaloPlex/NGS variants in the <i>GNAI3</i> gene.

<p>Primers used in Sanger sequencing for validation of identified HaloPlex/NGS variants in the <i>GNAI3</i> gene.</p

Validation of HaloPlex SNVs in the 5’ UTR of <i>GNAI3</i> by Sanger Sequencing.

<p><b>A.</b> Homozygous SNV (c.-72C>T) of Patient 2. At the top of the Figure the track annotation panel shows the Chr1 location of the C >T variant at position 109548649. Next, the forward sequences of the reference (R) and test sample (S) are shown, followed by the confidence score of the peak corresponding to the variant. From the bottom up, the reverse sequences of the reference and test sample peak profiles and the confidence score demonstrate the same variant. The red arrows point to the mutated nucleotide. <b>B.</b> Heterozygous SNV (c.-61C>T) of Patient 10. Same as <b>A,</b> except for the track annotation panel, which shows the C>T SNV at position 109548660 in Chr1.</p