Figure 3. 14-3-3ζ induces Lem8 to undergo self-cleavage.

(A) Self-processing of Lem8 requires 14-3-3ζ. His₆-Lem8 or His₆-Lem8_C280S was incubated with His₆-14-3-3ζ for 2 hr, proteins resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) were detected by Coomassie brilliant blue staining. The cysteine protease inhibitor E64 was added to the indicated samples at a final concentration of 10 μM. Similar results were obtained from at least three independent experiments and the data shown here were from one representative experiment. (B) The 14-3-3 protein from D. discoideum induces the self-cleavage of Lem8. His₆-Lem8 was incubated with GST-14-3-3ζ or GST-14-3-3Dd for the indicated time and the mixtures separated by SDS–PAGE were detected by immunoblotting with antibodies specific for Lem8 and GST, respectively. Similar results were obtained from at least three independent experiments and the data shown here were from one representative experiment. (C) Determination of the self-cleavage site of Lem8. His₆-Lem8 was incubated with His₆-14-3-3ζ for 16 hr, proteins were resolved by SDS–PAGE, stained with Coomassie brilliant blue. Protein bands corresponding to full-length and cleaved Lem8 band was excised, digested with trypsin and analyzed by mass spectrometry. The detection of the semitryptic peptide -L₄₆₄CEKAPQPTPQRQ₄₇₆- in cleaved samples suggested that the cleavage site lies between Gln476 and Arg477. (D) Mutations in cleavage site does not abolish Lem8 self-processing. Recombinant protein of Lem8 and the 4A mutant were each incubated with His₆-14-3-3ζ for 4 hr. Proteins resolved by SDS–PAGE were detected by Coomassie brilliant blue. Similar results were obtained from at least three independent experiments and the data shown here were from one representative experiment.

Figure 3—figure supplement 1. Identification of the self-cleavage sites of Lem8.

(A) Determination of the self-cleavage site of Lem8 by mass spectrometry. A diagram of the sequence containing the recognition site with the two diagnostic peptides used to determine the cleavage site (top panel). Protein bands corresponding to full-length and cleaved Lem8 band was excised (lower left panel), digested with trypsin and analyzed by mass spectrometry. The semitryptic peptide -L₄₆₄CEKAPQPTPQRQ₄₇₆- is present in cleaved samples but not in samples of full-length Lem8, whereas the fragment -A₄₇₈QSLSAETER₄₈₇- was only detected in samples of the full-length protein (lower right panel), supporting the notion that the cleavage site lies between Gln476 and Arg477 described in Figure 3C. (B) Self-cleavage of Lem8 removes GFP fused to its carboxyl end. GFP was fused to the indicated alleles of Lem8 and the fusion proteins were individually expressed in HEK293T cells by transfection. Samples resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) were detected by immunoblotting with GFP-specific antibodies. Results shown were one representative from three independent experiments with similar results. (C) The self-cleavage site of the 4A mutant. Protein bands from stained SDS–PAGE gels were excised and analyzed similarly as described in A. The tryptic fragment A₄₆₈PQPTPAAAAQSLSAETER₄₈₇- was detected only in samples prepared from the cleaved protein but not full-length protein. Identical results were obtained in multiple samples analyzed by two different mass spectrometry facilities.