Fig. 1

LMTK2 binds to both KLC1 and KLC2 via a C-terminal WD motif (a.a. 1388/1389). a LMTK2 binds to KLC1 and KLC2. HEK293 cells were transfected with either control empty vector (Ctrl), myc-LMTK2, FLAG-KLC1, FLAG-KLC2, myc-LMTK2 + FLAG-KLC1 or myc-LMTK2 + FLAG-KLC2. LMTK2, KLC1 and KLC2 were each immunoprecipitated via their epitope tags and bound KLC1/KLC2 or LMTK2 detected by immunoblotting. No signals for co-immunoprecipitated proteins were obtained in control cells transfected with only one plasmid which demonstrates the specificity of the assays. Both inputs and immunoprecipitates (IP) are shown. b and c Mutation of KLC1(N302) or KLC2(N287) to leucine within their TPR domains disrupts binding to LMTK2. HEK293 cells were transfected with control vector, myc-LMTK2 + FLAG-KLC1, myc-LMTK2 + FLAG-KLC1(N302L), myc-LMTK2 + FLAG-KLC2 or myc-LMTK2 + FLAG-KLC2(N287L). myc-LMTK2 was immunoprecipitated via the myc-tag and bound KLC1 (b) or KLC2 (c) detected on immunoblots. Mutant KLC1(N302L) did not bind to LMTK2 and KLC2(N287L) displayed reduced binding to LMTK2. Both inputs and immunoprecipitates are shown. Graph in (c) shows relative levels of KLC2 bound to LMTK2 in the immunoprecipitations following quantification of signals on immunoblots. Data were analysed by t-test. N = 5; error bars are s.e.m., *p < 0.05. Both inputs and immunoprecipitations (IP) are shown. d and e Mutation of LMTK2 WD (1388/1399) but not WE (477/478) to AA disrupts binding to KLC1 and KLC2. HEK293 cells were transfected with control vector, myc-LMTK2 + FLAG-KLC1/KLC2, myc-LMTK2(WE477/478) + FLAG-KLC1/KLC2 or myc-LMTK2(WD1388/1389) + KLC1/KLC2. LMTK2 was immunoprecipitated via its epitope tag and bound KLC1 (d) or KLC2 (e) detected by immunoblotting. Mutation of LMTK2 WD/AA (1388/1389) abolished binding of KLC1 and markedly inhibited binding of KLC2. Both inputs and immunoprecipitates are shown. Bar chart in (e) shows relative levels of KLC2 bound to LMTK2 in the immunoprecipitations following quantification of signals on immunoblots. Data were analysed by one-way ANOVA and Tukey’s post hoc test. N = 3; error bars are s.e.m., *p < 0.05.