Identification of KLC1ser460 as a phosphorylation site by LC–MS/MS. (A) A schematic representation of the KLC1 structure showing the heptad repeats (green box) and the TPR domain (comprising six tandem repeats numbered 1–6). Serine 460 (S460) is located in a linker region between TPR domains 5 and 6. (B,C) Tandem MS/MS spectra of the KLC1 phosphopeptide ACKVDSPTVTTTLK fragment ion series, after collision-induced dissociation. (B shows the spectrum with transfected FLAG–KLC1 and C shows that with endogenous KLC1 isolated from rat cortical neurons). The m/z of the fragment ions is plotted against the intensity, and the detected ions of the b- and y-ion collision series are indicated. The pattern of fragment ions produced localises the phosphorylation site in both samples to serine 460. (B) The mass difference between y₈ and y₉ and the release of phosphoric acid from y₉ (y₉-H₃PO₄) is consistent with a phosphorylation on serine 460. [M+2H]²⁺ represents the peptide ion with m/z=800.9, used as the parent ion for fragmentation to produce the MS/MS spectrum; [M+2H-H₃PO₄]²⁺ indicates the neutral loss of phosphoric acid. (C) Neutral loss from b₆ ([b -H₃PO₄]²⁺) but not from y₁ to y₈ localises the phosphorylation site to serine 460. Consecutively matched y-ions without neutral loss indicate no phosphorylation of other potential sites (threonine 462, threonine 464, threonine 465 and threonine 466). The parent ion is absent following the fragmentation. [M+3H-H₂O]³⁺ at m/z=528.12 and [M+3H-H₃PO₄]³⁺ at m/z=501.39 represent the loss of water and the neutral loss of phosphoric acid from the parent ion, respectively.

Mutation of KLC1ser460 to alanine or aspartate residues alters the binding of KLC1 to calsyntenin-1. (A) Cells were co-transfected with EGFP–calsyntenin-1 (calsyntenin) and either FLAG–KLC1wt, FLAG–KLC1ser460ala (KLC1ala) or FLAG–KLC1ser460asp (KLC1asp). KLC1 was immunoprecipitated (IP) using the anti-FLAG antibody and calsyntenin-1 was immunoprecipitated using the anti-GFP antibody. The samples were then probed on immunoblots for KLC1, using the anti-FLAG antibody, and for calsyntenin-1, using the anti-GFP antibody. To demonstrate the specificity of the immunoprecipitations, cells were also singly transfected with either FLAG–KLC1wt or EGFP–calsyntenin-1. Samples of the input lysates and immunoprecipitates are shown. In both immunoprecipitation assays, KLC1ser460ala bound approximately twofold more and KLC1ser460asp bound approximately twofold less calsyntenin-1 than KLC1wt. (B) Transfection of FLAG–KLC1 markedly increases KLC1 expression. Cells were transfected with empty vector (EV) or FLAG-tagged KLC1wt, KLC1ala or KLC1asp; total and transfected KLC1 was detected on immunoblots using anti-KLC1 and anti-FLAG antibodies as indicated. The samples were also probed for actin as a loading control. (C) siRNA-mediated knockdown of endogenous human but not transfected mouse FLAG–KLC1 in HEK-293 cells. Cells were co-transfected with either empty vector (EV) or FLAG–KLC1, and either control siRNA (Ctrl) or human-specific KLC1 siRNA as shown. Samples were probed on immunoblots for total KLC1 (KLC1) and transfected FLAG–KLC1 using the anti-FLAG antibody as indicated. The samples were also probed for actin as a loading control. (D) KLC1ser460ala binds approximately twice, and KLC1ser460asp binds approximately half, the calsyntenin-1 than KLC1wt following siRNA-mediated knockdown of endogenous KLC1. Cells were co-transfected with EGFP–calsyntenin-1 (calsyntenin) and human-specific KLC1 siRNA, and mouse FLAG–KLC1wt (KLC1wt), FLAG–KLC1ser460ala (KLC1ala) or FLAG–KLC1ser460asp (KLC1asp). KLC1 was immunoprecipitated using anti-FLAG antibody and the input and immunoprecipitate samples probed on immunoblots for KLC1, using the anti-FLAG antibody, and calsyntenin-1, using anti-GFP antibody.

Localisation of calsyntenin-1, and KLC1wt, KLC1ser460ala and KLC1ser460asp in transfected CV-1 cells. (A) Confocal images of cells transfected with EGFP–calsyntenin-1 and co-stained for PDI, GM130 or TGN-46. (B) Confocal images of CV1 cells transfected with FLAG–KLC1wt, FLAG–KLC1ser460ala (KLC1ala) or FLAG–KLC1ser460asp (KLC1asp). Scale bars: 20 μm.

Mutation of KLC1ser460 alters axonal transport of calsyntenin-1. Rat cortical neurons were co-transfected with EGFP–calsyntenin-1 and FLAG–KLC1wt (KLC1wt), FLAG–KLC1ser460ala (KLC1ala) or FLAG–KLC1ser460asp (KLC1asp), and calsyntenin-1 transport was recorded in time-lapse through visualisation of the EGFP tag. (A) Representative kymographs for each transfection are shown. (B,C) Histograms showing the number (expressed as a normalised percentage) of EGFP–calsyntenin-1 particles moving in both anterograde and retrograde directions in the presence of KLC1wt, KLC1ala or KLC1asp. KLC1ala increases, whereas KLC1asp decreases, anterograde movement, compared with that in KLC1wt; corresponding changes in retrograde movements are observed. Data were analysed by one-way ANOVA with LSD post-hoc test (*P<0.05; **P<0.001) for n=9 cells for each transfection from three independent experiments. Error bars are s.e.m.

Mutation of KLC1ser460 into alanine or aspartate residues does not affect the binding of KLC1 to KHC or to HAP1A, CRMP2, JIP1 or Kidins220. (A) CHO cells were transfected with FLAG–KLC1wt, FLAG–KLC1ser460ala (KLC1ala), FLAG–KLC1ser460asp (KLC1asp) or empty vector (EV), and KLC1 was immunoprecipitated (IP) using the anti-FLAG antibody. Samples were probed on immunoblots for KLC1, using anti-FLAG antibody, and for endogenous KHC, using the antibody pcp42. (B–E) CHO cells were co-transfected with FLAG–KLC1wt, FLAG–KLC1ser460ala or FLAG–KLC1ser460asp and Myc–HAP1A (B), Myc–CRMP2 (C), Myc–JIP1 (D) or EGFP–Kidins220 (E). KLC1 was immunoprecipitated using the anti-FLAG antibody. The samples were probed on immunoblots for KLC1, using anti-FLAG antibody, and bound cargoes, using anti-Myc or anti-GFP antibodies. To demonstrate the specificity of the immunoprecipitations, cells were also singly transfected with KLC1wt, or with Myc–HAP1A (B), Myc–CRMP2 (C), Myc–JIP1 (D) or EGFP–Kidins220 (E). Samples of the input lysates and immunoprecipitates are shown.

KLC1ser460 is phosphorylated by ERK and inhibition of ERK increases binding of KLC1 to calsyntenin-1. (A) Inhibition of ERK promotes binding of calsyntenin-1 to KLC1wt but has no effect on its binding to KLC1ser460ala in immunoprecipitation (IP) assays from co-transfected CHO cells. Cells were co-transfected with EGFP–calsyntenin-1 (calsyntenin) and either FLAG–KLC1wt (KLC1wt) or FLAG-KLC1ser460ala (KLC1ala), and the cells were then treated (+) or not treated (−) with U0126 to inhibit ERK. KLC1 was immunoprecipitated using the anti-FLAG antibody and the samples were probed on immunoblots for KLC1, using the anti-FLAG antibody, and calsyntenin-1, using the anti-GFP antibody. Samples of the input lysates and immunoprecipitates are shown. Also shown are immunoblots for total and active ERK (T-ERK and P-ERK, respectively). (B,C) Inhibition of JNK (B) or p38 (C) does not influence binding of KLC1 to calsyntenin-1 in immunoprecipitation assays from co-transfected CHO cells. Cells were co-transfected with EGFP–calsyntenin-1 and FLAG–KLC1wt, and the cells were then treated (+) or not treated (−) with SP600125, to inhibit JNK (B), or SB203580, to inhibit p38 (C). KLC1 was immunoprecipitated using the anti-FLAG antibody, and the samples were probed on immunoblots for KLC1, using the anti-FLAG antibody, and calsyntenin-1, using the anti-GFP antibody. Samples of the input lysates and immunoprecipitates are shown. Also shown are immunoblots for total JNK (p54 isoform) and phosphorylated ATF2 (P-ATF2) in (B), to show JNK inhibition, and total p38 and phosphorylated MSK1 (P-MSK1) in (C), to show MAPK inhibition. ATF2 and MSK1 are downstream substrates for JNK and p38, respectively. (D) In vitro phosphorylation of KLC1(154–534) but not KLC1(154–534)ser460ala by ERK. KLC1wt or KLC1ser460ala substrates were prepared and phosphorylated in vitro with [γ-³²P]ATP and ERK. RM is reaction mix only with no substrate; − and + refer to the presence or absence of ERK in the reaction. The upper panel is a Coomassie-Blue-stained gel to show KLC1 substrates; the lower panel is the autoradiograph. (E) Inhibition of ERK promotes binding of endogenous calsyntenin-1 to endogenous kinesin-1 motors in immunoprecipitation assays from 7DIV rat cortical neurons. Kinesin-1 was immunoprecipitated using the SUK4 antibody, and bound KLC1 and calsyntenin-1 were detected on immunoblots using rabbit anti-KLC antibody and anti-calsyntenin-1 antibody, respectively; KHC was detected using H2. Control immunoprecipitations (Ctrl) were performed using non-immune mouse Igs. Samples of the input lysates and immunoprecipitates are shown. Also shown are immunoblots for total and active ERK; − and + represent absence or presence of U0126 treatment.

Mutation of KLC1ser460 alters KLC1 co-localisation with calsyntenin-1 in transfected CV-1 cells. CV-1 cells were co-transfected with EGFP–calsyntenin-1 and FLAG–KLC1wt, FLAG–KLC1ser460ala (KLC1ala) or FLAG–KLC1ser460asp (KLC1asp). Calsyntenin-1 was detected through the EGFP tag and KLC1 using the anti-FLAG antibody; images shown are confocal sections. (A) Calsyntenin-1 is highly co-localised with wild-type and both mutant KLC1s in perinuclear regions. In addition calsyntenin-1 and FLAG–KLC1ser460ala co-localise in plasma membrane projections; such co-staining of projections was less commonly seen in calsyntenin-1 plus FLAG–KLC1ser460asp co-transfected cells (arrowheads). Magnified areas of interest are shown, with co-localised pixels shown in the lower zoom panel. (B) ICQ values for colocalisation of calsyntenin-1 with KLC1wt, KLC1ser460ala or KLC1ser460asp. Calsyntenin-1 with KLC1wt, ICQ 0.33±0.04 (n=10); calsyntenin-1 with KLC1ser460ala, ICQ 0.394±0.05 (n=10); calsyntenin-1 with KLC1ser460asp: ICQ 0.225±0.05 (n=10). Data were analysed by one-way ANOVA with a LSD post-hoc test (*P<0.05; **P<0.01). Values are means ± s.d. Scale bars: 20 μm (2.5 μm in zoom panels).