Figure 5.

JNK Phosphorylates Syt4 at S135 to Promote DCV Capture

(A and B) Western blot of co-immunoprecipitation of FLAG-JNK and GFP-Syt4 co-expressed in HEK cells, using anti-FLAG beads (A, +), anti-GFP beads (B, +), or control beads without antibody (−). Input, IP, and S are indicated.

(C) GPS kinase prediction, where a score/cutoff > 2 is positive.

(D) In vitro kinase assay quantitation of percent JNK1-dependent phosphorylation of WT and phosphodeficient peptides of Syt4 and c-Jun.

(E) Color-coded vesicle tracks (top; scale bar, 10 μm) and kymographs (bottom; scale bar, 5 μm) from hippocampal neurons transfected with mCherry-tagged Syt4 alone (control) or co-expressed with dominant-negative JNK1 (JNK1(APF)) or overexpressed active JNK1 (MKK7-JNK1) and S135A Syt4 with overexpressed JNK1 (S135A/MKK7-JNK1). Scale bar, 10 μm.

(F–I) Quantitation of velocity (F), mobile percentage (G), pause frequency (H), and pause time (I) of Syt4 vesicles under JNK1(APF), MKK7-JNK1, and S135A/MKK7-JNK1 conditions, normalized to WT control (n = 5,216, 7,478, 3,172, and 4,232, vesicles for velocity, pause frequency, and pause time and n = 22, 27, 27, and 18 movies for mobile vesicle percentage for control, JNK1(APF), MKK7-JNK1, and S135A/MKK7-JNK1, respectively, from 4 cultures).

(J) Axons of neurons co-transfected with syp-GFP and mCherry-Syt4, mCherry-Syt4-P2A-JNK1(APF), mCherry-Syt4-P2A-MKK7-JNK1, or mCherry-Syt4(S135A)-P2A-MKK7-JNK1. Scale bar, 10 μm.

(K) Quantitation of the percentage of mCherry-Syt4 vesicles co-localized with syp-GFP (n = 12, 17, and 21 cells for control, JNK1(APF), and MKK7-JNK1, respectively, from 3 cultures).

Significance was determined by Student’s t test with Bonferroni correction; error is SEM; ^∗∗p < 0.01, ^∗∗∗p < 0.001.