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. 1993 Dec 1;296(Pt 2):409–415. doi: 10.1042/bj2960409

The 50 kDa protein subunit of assembly polypeptide (AP) AP-2 adaptor from clathrin-coated vesicles is phosphorylated on threonine-156 by AP-1 and a soluble AP50 kinase which co-purifies with the assembly polypeptides.

A Pauloin 1, C Thurieau 1
PMCID: PMC1137711  PMID: 8257432

Abstract

AP50 is a subunit of the assembly polypeptide (AP) subclass AP-2 from bovine brain coated vesicles. It can be phosphorylated in vivo and in vitro on a threonine residue by means of the AP50 kinase activity associated with AP. We have undertaken an analysis of the amino acid sequence around the AP50 phosphorylation site. After phosphorylation in vitro of AP50 followed by tryptic cleavage, only one radioactive peptide was isolated following Mono-Q ion-exchange f.p.l.c. and reverse-phase h.p.l.c. The amino acid sequence of this peptide: Glu146-Glu-Gln-Ser-Gln-Ile-Thr-Ser-Gln-Val-Thr*-Gly-Gly-Ile-Gly-Tr p-Arg162, displayed two threonine residues. Analysis of the yield and radioactivity of the product from automated Edman degradation indicated that only Thr-156 was phosphorylated, reflecting the presence of a single phosphorylation site in AP50. AP phosphorylated the corresponding synthetic peptide on the same threonyl residue. We demonstrated that AP50 was a phosphorylation substrate unable to autophosphorylate. The enzyme involved in the AP50 phosphorylation was shown to be associated with AP-1 and with a soluble protein complex co-purified with APs but resolved from the latter by hydroxyapatite-column exclusion chromatography. This AP50 kinase activity corresponded to a 280 kDa protein complex according to gel-filtration data.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Ahle S., Mann A., Eichelsbacher U., Ungewickell E. Structural relationships between clathrin assembly proteins from the Golgi and the plasma membrane. EMBO J. 1988 Apr;7(4):919–929. doi: 10.1002/j.1460-2075.1988.tb02897.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bar-Zvi D., Branton D. Clathrin-coated vesicles contain two protein kinase activities. Phosphorylation of clathrin beta-light chain by casein kinase II. J Biol Chem. 1986 Jul 25;261(21):9614–9621. [PubMed] [Google Scholar]
  3. Bar-Zvi D., Mosley S. T., Branton D. In vivo phosphorylation of clathrin-coated vesicle proteins from rat reticulocytes. J Biol Chem. 1988 Mar 25;263(9):4408–4415. [PubMed] [Google Scholar]
  4. Beck K. A., Keen J. H. Self-association of the plasma membrane-associated clathrin assembly protein AP-2. J Biol Chem. 1991 Mar 5;266(7):4437–4441. [PubMed] [Google Scholar]
  5. Cantournet B., Creuzet C., Komano O., Loeb J. Clathrin beta-light chain of rat liver coated vesicles is phosphorylated in vitro and in vivo. FEBS Lett. 1987 Aug 10;220(1):143–148. doi: 10.1016/0014-5793(87)80892-x. [DOI] [PubMed] [Google Scholar]
  6. Glickman J. N., Conibear E., Pearse B. M. Specificity of binding of clathrin adaptors to signals on the mannose-6-phosphate/insulin-like growth factor II receptor. EMBO J. 1989 Apr;8(4):1041–1047. doi: 10.1002/j.1460-2075.1989.tb03471.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Goldstein J. L., Brown M. S., Anderson R. G., Russell D. W., Schneider W. J. Receptor-mediated endocytosis: concepts emerging from the LDL receptor system. Annu Rev Cell Biol. 1985;1:1–39. doi: 10.1146/annurev.cb.01.110185.000245. [DOI] [PubMed] [Google Scholar]
  8. Hager D. A., Burgess R. R. Elution of proteins from sodium dodecyl sulfate-polyacrylamide gels, removal of sodium dodecyl sulfate, and renaturation of enzymatic activity: results with sigma subunit of Escherichia coli RNA polymerase, wheat germ DNA topoisomerase, and other enzymes. Anal Biochem. 1980 Nov 15;109(1):76–86. doi: 10.1016/0003-2697(80)90013-5. [DOI] [PubMed] [Google Scholar]
  9. Keen J. H., Black M. M. The phosphorylation of coated membrane proteins in intact neurons. J Cell Biol. 1986 Apr;102(4):1325–1333. doi: 10.1083/jcb.102.4.1325. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Keen J. H., Chestnut M. H., Beck K. A. The clathrin coat assembly polypeptide complex. Autophosphorylation and assembly activities. J Biol Chem. 1987 Mar 15;262(8):3864–3871. [PubMed] [Google Scholar]
  11. Keen J. H. Clathrin assembly proteins: affinity purification and a model for coat assembly. J Cell Biol. 1987 Nov;105(5):1989–1998. doi: 10.1083/jcb.105.5.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Keen J. H., Willingham M. C., Pastan I. H. Clathrin-coated vesicles: isolation, dissociation and factor-dependent reassociation of clathrin baskets. Cell. 1979 Feb;16(2):303–312. doi: 10.1016/0092-8674(79)90007-2. [DOI] [PubMed] [Google Scholar]
  13. Kemp B. E., Benjamini E., Krebs E. G. Synthetic hexapeptide substrates and inhibitors of 3':5'-cyclic AMP-dependent protein kinase. Proc Natl Acad Sci U S A. 1976 Apr;73(4):1038–1042. doi: 10.1073/pnas.73.4.1038. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kemp B. E., Pearson R. B. Protein kinase recognition sequence motifs. Trends Biochem Sci. 1990 Sep;15(9):342–346. doi: 10.1016/0968-0004(90)90073-k. [DOI] [PubMed] [Google Scholar]
  15. Kennelly P. J., Krebs E. G. Consensus sequences as substrate specificity determinants for protein kinases and protein phosphatases. J Biol Chem. 1991 Aug 25;266(24):15555–15558. [PubMed] [Google Scholar]
  16. Kirchhausen T., Nathanson K. L., Matsui W., Vaisberg A., Chow E. P., Burne C., Keen J. H., Davis A. E. Structural and functional division into two domains of the large (100- to 115-kDa) chains of the clathrin-associated protein complex AP-2. Proc Natl Acad Sci U S A. 1989 Apr;86(8):2612–2616. doi: 10.1073/pnas.86.8.2612. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  18. Lees-Miller S. P., Anderson C. W. The human double-stranded DNA-activated protein kinase phosphorylates the 90-kDa heat-shock protein, hsp90 alpha at two NH2-terminal threonine residues. J Biol Chem. 1989 Oct 15;264(29):17275–17280. [PubMed] [Google Scholar]
  19. Lemmon S. K., Pellicena-Palle A., Conley K., Freund C. L. Sequence of the clathrin heavy chain from Saccharomyces cerevisiae and requirement of the COOH terminus for clathrin function. J Cell Biol. 1991 Jan;112(1):65–80. doi: 10.1083/jcb.112.1.65. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Manfredi J. J., Bazari W. L. Purification and characterization of two distinct complexes of assembly polypeptides from calf brain coated vesicles that differ in their polypeptide composition and kinase activities. J Biol Chem. 1987 Sep 5;262(25):12182–12188. [PubMed] [Google Scholar]
  21. Merril C. R., Goldman D., Sedman S. A., Ebert M. H. Ultrasensitive stain for proteins in polyacrylamide gels shows regional variation in cerebrospinal fluid proteins. Science. 1981 Mar 27;211(4489):1437–1438. doi: 10.1126/science.6162199. [DOI] [PubMed] [Google Scholar]
  22. Murphy J. E., Pleasure I. T., Puszkin S., Prasad K., Keen J. H. Clathrin assembly protein AP-3. The identity of the 155K protein, AP 180, and NP185 and demonstration of a clathrin binding domain. J Biol Chem. 1991 Mar 5;266(7):4401–4408. [PubMed] [Google Scholar]
  23. Nandi P. K., Irace G., Van Jaarsveld P. P., Lippoldt R. E., Edelhoch H. Instability of coated vesicles in concentrated sucrose solutions. Proc Natl Acad Sci U S A. 1982 Oct;79(19):5881–5885. doi: 10.1073/pnas.79.19.5881. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Pauloin A., Bernier I., eJollès P. Presence of cyclic nucleotide-Ca2+ independent protein kinase in bovine brain coated vesicles. Nature. 1982 Aug 5;298(5874):574–576. doi: 10.1038/298574a0. [DOI] [PubMed] [Google Scholar]
  25. Pauloin A., Jollès P. Internal control of the coated vesicle pp50-specific kinase complex. Nature. 1984 Sep 20;311(5983):265–267. doi: 10.1038/311265a0. [DOI] [PubMed] [Google Scholar]
  26. Pauloin A., Jollès P. Presence of a MgATP/ADP-dependent pp50 phosphatase in bovine brain coated vesicles. J Biol Chem. 1986 Sep 25;261(27):12568–12573. [PubMed] [Google Scholar]
  27. Pauloin A., Loeb J., Jollés P. Protein kinase(s) in bovine brain coated vesicles. Biochim Biophys Acta. 1984 Jun 29;799(3):238–245. doi: 10.1016/0304-4165(84)90266-6. [DOI] [PubMed] [Google Scholar]
  28. Pauloin A., Thurieau C., Jollès P. Cyclic phosphorylation/dephosphorylation cascade in bovine brain coated vesicles. Biochim Biophys Acta. 1988 Jan 18;968(1):91–95. doi: 10.1016/0167-4889(88)90048-1. [DOI] [PubMed] [Google Scholar]
  29. Pearse B. M. Coated vesicles from pig brain: purification and biochemical characterization. J Mol Biol. 1975 Sep 5;97(1):93–98. doi: 10.1016/s0022-2836(75)80024-6. [DOI] [PubMed] [Google Scholar]
  30. Pearse B. M. Receptors compete for adaptors found in plasma membrane coated pits. EMBO J. 1988 Nov;7(11):3331–3336. doi: 10.1002/j.1460-2075.1988.tb03204.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Pearse B. M., Robinson M. S. Purification and properties of 100-kd proteins from coated vesicles and their reconstitution with clathrin. EMBO J. 1984 Sep;3(9):1951–1957. doi: 10.1002/j.1460-2075.1984.tb02075.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Peterson G. L. A simplification of the protein assay method of Lowry et al. which is more generally applicable. Anal Biochem. 1977 Dec;83(2):346–356. doi: 10.1016/0003-2697(77)90043-4. [DOI] [PubMed] [Google Scholar]
  33. Pfeffer S. R., Rothman J. E. Biosynthetic protein transport and sorting by the endoplasmic reticulum and Golgi. Annu Rev Biochem. 1987;56:829–852. doi: 10.1146/annurev.bi.56.070187.004145. [DOI] [PubMed] [Google Scholar]
  34. Robinson M. S. 100-kD coated vesicle proteins: molecular heterogeneity and intracellular distribution studied with monoclonal antibodies. J Cell Biol. 1987 Apr;104(4):887–895. doi: 10.1083/jcb.104.4.887. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Robinson M. S. Cloning of cDNAs encoding two related 100-kD coated vesicle proteins (alpha-adaptins). J Cell Biol. 1989 Mar;108(3):833–842. doi: 10.1083/jcb.108.3.833. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Soderling T. R. Protein kinases. Regulation by autoinhibitory domains. J Biol Chem. 1990 Feb 5;265(4):1823–1826. [PubMed] [Google Scholar]
  37. Thurieau C., Brosius J., Burne C., Jolles P., Keen J. H., Mattaliano R. J., Chow E. P., Ramachandran K. L., Kirchhausen T. Molecular cloning and complete amino acid sequence of AP50, an assembly protein associated with clathrin-coated vesicles. DNA. 1988 Dec;7(10):663–669. doi: 10.1089/dna.1988.7.663. [DOI] [PubMed] [Google Scholar]
  38. Ungewickell E., Branton D. Assembly units of clathrin coats. Nature. 1981 Jan 29;289(5796):420–422. doi: 10.1038/289420a0. [DOI] [PubMed] [Google Scholar]
  39. Zaremba S., Keen J. H. Assembly polypeptides from coated vesicles mediate reassembly of unique clathrin coats. J Cell Biol. 1983 Nov;97(5 Pt 1):1339–1347. doi: 10.1083/jcb.97.5.1339. [DOI] [PMC free article] [PubMed] [Google Scholar]

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