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. 2016 Jul 28;5:e17159. doi: 10.7554/eLife.17159

Figure 5. Prmt1 binds to and methylates KCNQ2 at R333, R345, R353, and R435.

(a–c) Immunoblotting analysis showing the physical association of KCNQ2 and Prmt1. HEK293T cells were transfected with expression vectors, as indicated. Whole-cell lysates were immunoprecipitated and immunoblotted by either anti-flag or anti-HA antibody. Representative data from at least three independent experiments are shown. (d) Western blotting analysis showing endogenous interaction of KCNQ2 and Prmt1. Cell lysates from mouse hippocampus were immunoprecipitated with anti-Prmt1 antibody and were immunoblotted with anti-KCNQ2 antibody or anti-Prmt1 antibody. Representative data from at least three independent experiments are shown. (e) In vitro methylation assays with GST-KCNQ2 (amino acids 320–449) and a series of GST-Prmts (1–6) in the presence of [3H]S-adenosylmethionine (SAM). Total amounts of GST-KCNQ2 (arrowhead) and GST-Prmts are shown by Coomassie brilliant blue staining. (f) In vitro methylation assays with GST-Prmt1 together with GST-KCNQ2 (amino acids 320–449) WT, R333K/R353K, R345K/R353K R345K/R435K. R333K/R345K/R353K (RRRR-KKKR), or R333K/R345K/R353K/R435K (4RK) in the presence of [3H]SAM. (g) Immunoblotting analysis showing the decreased asymmetric dimethylation of KCNQ2 in Prmt1 knockdown cells. HEK293T cells were transfected with control or HA-KCNQ2 in combination with control or Prmt1 shRNA expression vectors, followed by immunoprecipitation with KCNQ2 antibodies and immunoblotting with antibodies to ADMA, KCNQ2, Prmt1 and GAPDH. (h) Immunoblotting analysis showing the enhanced asymmetric dimethylation of KCNQ2 in Prmt1 overexpressing cells. HEK293T cells were transfected with control or HA-KCNQ2 in combination with control or Prmt1 expression vectors, followed by immunoprecipitation with KCNQ2 antibodies. (i) Immunoblotting analysis showing decreased asymmetric dimethylation of KCNQ2 in Prmt1+/- brain, compared to the WT control. Brain lysates from Prmt1+/+ and Prmt1+/- mice were immunoprecipitated with anti-KCNQ2 antibodies or control IgG.

DOI: http://dx.doi.org/10.7554/eLife.17159.013

Figure 5.

Figure 5—figure supplement 1. Expression of KCNQ2 and KCNQ3 in the hippocampus of WT and Prmt1+/- mice.

Figure 5—figure supplement 1.

DOI: http://dx.doi.org/10.7554/eLife.17159.014
Figure 5—figure supplement 2. Identification of in vivo arginine methylation sites of KCNQ2 MS/MS spectra of the methylated peptides.

Figure 5—figure supplement 2.

(a) 332RRNPAAGLIQSAWR345, (b) 334NPAAGLIQSAWR345, (c) 346FYATNLSR353 and (d) 435RSPSADQSLEDSPSK449. R* represents monomethylated arginine and R** represents dimethylated arginine.
DOI: http://dx.doi.org/10.7554/eLife.17159.015
Figure 5—figure supplement 3. The sequence alignment of 5 difference human KCNQ isoforms.

Figure 5—figure supplement 3.

The arginine residues critical for methylation in KCNQ2 are marked in red. KCNQ3 and KCNQ4 have 3 conserved arginine residues while KCNQ5 and KCNQ1 have two or one conserved arginine residues, respectively. Note that KCNQ2 and KCNQ3 form M currents in brain.
DOI: http://dx.doi.org/10.7554/eLife.17159.016
Figure 5—figure supplement 4. In vitro methylation assays with GST-Prmt1 or myc-Prmt5 together with GST-KCNQ2 (320-449 aa).

Figure 5—figure supplement 4.

In vitro methylation assays with myc-Prmt5 purified from HEK293T cells and GST-Prmt1 by using GST-KCNQ2 (aa 320–449) as a substrate and histone as a positive control. Coomassie blue stainings show the loading controls for each protein.
DOI: http://dx.doi.org/10.7554/eLife.17159.017
Figure 5—figure supplement 5. Expression of Prmt8 in Prmt1+/+ and Prmt1+/- brain lysates. .

Figure 5—figure supplement 5.

DOI: http://dx.doi.org/10.7554/eLife.17159.018