The phosphorylation-dependent binding of 14-3-3s to kinesin light chain isoforms. A, To identify in vitro AMPK-phosphorylated sites on GST-KLC2 that promote binding to 14-3-3s, GST-KLC2 was phosphorylated by AMPK in vitro with Mg[γ32P]ATP. A sample was checked for binding to 14-3-3 in a Far Western assay (similar to B. WT), and the remainder digested with trypsin and run on HPLC with the indicated gradient of acetonitrile in 0.1% trifluoroacetic acid. The 32P trace shows three phosphorylated peptides P1, P2, and P3, which were identified by mass spectrometry to be singly phosphorylated peptides with the amino acid sequences shown. The recovery of 32P from HPLC was 87%. The phosphorylated residue in P3 was not pinpointed in this experiment, but other data (see text) are consistent with it being Ser582. The bottom panel shows that a release of radioactive inorganic phosphate at the third cycle of solid-phase Edman degradation of P1 revealed Ser545 as the phosphorylated residue in that peptide (performed as in ()). B, 14-3-3 binding to GST-KLC2 phosphorylated in vitro with AMPK and the effects of Ala and Asp substitutions of Ser residues. Proteins were incubated with AMPK with and without MgATP, and subject to 14-3-3 Far Western binding assay, and immunoblotting with antiphosphoSer545 and antiphosphoSer582, with anti-GST as loading control. In addition to the phosphoSer545 and phosphoSer582 14-3-3 binding sites identified here (see text), mutations of the adjacent Ser581 and also Ser428 (reported to be phosphorylated (http://www.phosphosite.org)) and within a Rxx(pS)xP sequence that looks like a 14-3-3-binding motif) were tested, giving no indication that these sites are involved in 14-3-3 binding. As an aside, note that in contrast to 14-3-3, the pSer582 antibody recognizes KLC2 with Asp as a phosphomimetic in place of pSer582. In contrast, the antiphosphoSer545 antibody does not recognize Asp545. Also, mutation of the neighboring residue Ser581 to Ala reduces the ability of the pSer582 antibody, but not 14-3-3, to recognize pSer582. C, HA-KLC2 was isolated from transfected HEK293 cells that were serum deprived for 5 h, incubated with or without H-89 (30 μm for 30 min), and stimulated with the adenylate cyclase activator forskolin (20 μm for 30 min). The HA-KLC2 was probed for binding to antiphosphoSer545-KLC2, antiphosphoSer582-KLC2 and DIG-14-3-3. The PKA-phosphorylation of Ser157 on VASP was monitored to assess the activation of PKA. D, Wild-type and Ser545Ala/Ser582Ala forms of GFP-KLC2 were isolated from lysates of transfected HEK293 cells in the presence and absence of 2.5 mg/ml dithiobis[succinimidyl propionate] (DSP) to stabilize any labile interactions and assayed for direct binding to 14-3-3 in a Far Western, and for the presence of associated proteins with the indicated antibodies. E, C-terminal sequences of KLC2 from various species aligned, together with human KLC3 and KLC4 and the C-terminal splice variants of human KLC1 (KNS2 gene; Ensembl ENST00000303439) as reported previously (). The asterisks align with the positions shaded in gray of Ser545 and Ser582 in human KLC2. Residues in lowercase letters have been shown to be phosphorylated here and elsewhere (http://www.phosphosite.org). Underlined residues (Ser611, Ser615) on mouse KLC2 were proposed as GSK3 sites (); with the preceding bold S proposed as a priming site, though we note that Ser615 has not been shown to be phosphorylated and our attempts to phosphorylate KLC2 with GSK3 in vitro and in vivo, in experiments using priming kinases and GSK3 inhibitors, were unsuccessful (not shown, including experiments with Adam Cole, University of Dundee). Accession numbers are: hKLC1 Q07866 (human), hKLC2 Q9H0B6 (human), hKLC3 Q6P597 (human), hKLC4 Q9NSK0 (human), mKLC2 O88448 (mouse) and dKLC1 Q7ZVT4 (Zebra fish, Danio rerio). F, Wild type and mutant HA-tagged KLC1 and KLC2 proteins were immunoprecipitated from HEK293 cells probed with antiphosphoSer545-KLC2, antiphosphoSer582-KLC2 and by Far Western with DIG-14-3-3.