SEQUENCE OF SITES ON ATP-CITRATE LYASE AND PHOSPHATASE INHIBITOR-2 PHOSPHORYLATED BY MULTIFUNCTIONAL PROTEIN-KINASE (A GLYCOGEN-SYNTHASE KINASE-3 LIKE KINASE)

Multifunctional protein kinase (MFPK) phosphorylates ATP-citrate lyase on peptide B on two sites, BT and BS, on threonine and serine, respectively, inhibitor 2 on a threonyl residue, and glycogen synthase at sites 2 and 3. The phosphorylation sites BT and BS of ATP-citrate lyase are dependent on prior phosphorylation at site A whereas site A phosphorylation is decreased by prior phosphorylation at sites BT and BS. To study the MFPK recognition sites and the site-site interactions, the amino acid sequences of ATP-citrate lyase peptide B and inhibitor 2 were determined and compared to each other and to glycogen synthase sites 3-5. The sequence of the tryptic peptide containing the two phosphorylation sites of peptide B is -Phe-Leu-Leu-Asn-Ala-Ser-Gly-Ser-Thr-Ser-Thr(P)-Pro-Ala-Pro-Ser(P)-Arg-, and the sequence of the MFPK phosphorylation site of inhibitor 2 is -Ile-Asp-Glu-Pro-Ser-Thr(P)-Pro-Tyr-. This inhibitor 2 site is identical with the site phosphorylated by glycogen synthase kinase 3/FA. These results suggest that at least some of the sites phosphorylated by MFPK (BT of ATP-citrate lyase, Thr 72 of inhibitor 2, and sites 3b and 4 of glycogen synthase) contain a Ser/Thr flanked by a carboxyl-terminal proline. However, as MFPK did not phosphorylate a series of peptides containing the -X-Thr/Ser-Pro-X- sequence, this minimum consensus sequence is not sufficient for phosphorylation by MFPK. MFPK was able to phosphorylate glycogen synthase synthetic peptide only after it had first been phosphorylated by casein kinase II at site 5, supporting the hypothesis that the phosphorylation of natural substrates by MFPK depends on prior phosphorylation at other sites. ATP-citrate lyase sites BT and BS resemble glycogen synthase sites 3b and 3c, suggesting that MFPK recognizes a Ser/Thr in the following pattern: -Ser/Thr()-X-X-X-Ser/Thr(P)-. Additional evidence for this four amino acid spacing was obtained when the sequence about all three phosphorylation sites of ATP-citrate lyase was found to be-Thr(P)-Pro-Ala-Pro-Ser(P)-Arg- Thr-Ala-Ser(P)-, a configuration that resembles glycogen synthase sites 3b, 3c, and 4. © 1990, American Chemical Society. All rights reserved.