Figure 1.

The atypical domains of MyoK are functional. (A) MyoK contains a unique GPR domain (aa 122-265; pale blue) inserted in loop 1 of the motor domain. No neck, no light chain binding site, and no polybasic stretch can be recognized after the last conserved amino acids of the motor domain (KIF; aa 806-808). Instead, the atypical short tail (magenta) ends with a CAAX box (CLIQ; aa 855-858). (B) In the sequence of the GPR loop, the type I SH3 binding motif is boxed in green. The putative profilin binding poly-proline stretch is boxed in red. Unconventional profilin binding motifs, ZPPϕ (where Z is generally proline, glycine, or alanine and ϕ a hydrophobic residue) are boxed in black. (C) GST and GST-GPR coupled beads were incubated with wild-type cytosol and eluted with 0.1 mM PLP, showing that profilin-actin quantitatively dissociated from GST-GPR but not GST. (D) GST-GPR coupled beads were incubated with wild-type cytosol and eluted with 100 μM PLP and 1 M NaCl. Eluates were electrophoresed and gels Coomassie stained. Asterisks indicate two major unidentified protein bands. (E) MyoK and PakB were coimmunoprecipitated with anti-Abp1 antibodies. (F) MyoK is phosphorylated in vivo at the TEDS site, as revealed by the fragmentation spectrum of the peptide HTQYQVPQNPDQSAGLRDALAK that displays the y18-y21 sequence ions (2+) that identify the N-terminal TYQ amino acids (box) together with a neutral loss of H₃PO₄, characteristic of phosphorylated threonine. Peaks corresponding to H₂O loss are indicated by stars. The phosphorylated threonine is in red and conserved residues neighboring the TEDS site are underlined. (G and H) MyoK, MyoD but not MyoB are phosphorylated in vitro by PakB. (G) The autoradiograms show the incorporation of ³²P into purified myosin head constructs at indicated times of incubation with [γ-³²P]ATP and purified GST-PakB. (H) The rates of phosphate incorporation were linear up to 60 min for all reactions. Data are representative of three independent assays. (I) The GFP-MyoK tail construct localized to the plasma membrane (arrow). It was not excluded from the phagocytic cup but progressively lost during maturation (arrowheads). The nucleus is indicated by an asterisk. Bar, 5 μm.