Skip to main content
SpringerLink
Log in

Cloning and expression of a new human polypeptide which regulates protein phosphorylation in Escherichia coli

  • Published:
Molecular and Cellular Biochemistry Aims and scope Submit manuscript

Summary

A 1,820bp full-length clone encoding for a new human protein was isolated from a λgt11 placental cDNA library using anti-human hexokinase antibodies. The cDNA complete sequence includes a 12 by 5′ noncoding region, a single open reading frame encoding a protein of 55 KDa (HP-10) and a 177 by non-coding with two putative polyadenylation signals upstream of 3′poly(A)tail. The deduced amino acid sequence reveals a sequence of 492 amino acids that contains a stretch of 7 glutamic acid from position 169 and one potential glycosylation site at position 274. Although antibodies against hexokinase recognize the fusion protein and antibodies against the fusion protein recognize hexokinase, HP-10 is not human hexokinase, by a number of criteria including the alignment of determined amino acid sequences.

In searching for a possible functional role of HP-10 its cDNA was inserted into a procaryotic vector which allows the expression of the non-fused protein. Bacteria expressing the HP-10 encoded protein were isolated and found to have a dramatic increase in endogenous phosphorylated proteins. Since HP-10 does not have a protein kinase activity per se it should be considered a new regulatory phosphorylation protein which is active in E. coli

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

Abbreviations

HK:

Hexokinase (EC 2.7.1.1)

References

  1. Edelman AM, Blumenthal DK, Krebs EG: In: CC Riehardson, PD Boyer, IB David, A Meister (eds.) Protein serine/threonine kinases. Ann Rev Biochem. Annual Reviews Inc, Palo Alto, vol. 56, 1987, pp 567–613

    Google Scholar 

  2. Krebs EG: In: PD Boyer (ed.) The enzymes. Academic Press, New York, vol 17, 1986, pp 3–18

    Google Scholar 

  3. McVey D, Brizuela L, Mohr I, Marshak DR, Glizman Y, Beach D: Phosphorylation of large tumor antigen by cdc2 stimulates SV40 DNA replication, Nature 341: 503–507, 1989

    CAS  PubMed  Google Scholar 

  4. Murray AV: The cell cycle as a cdc 2 cycle. Nature 342: 14–15, 1989

    Google Scholar 

  5. Gould KL, Nurse P: Tyrosine phosphorylation of the fission yeast cdc2+ protein kinase regulates entry into mitosis. Nature 342: 39–45, 1989

    Google Scholar 

  6. Varms H: The molecular basis of blood diseases. In: G Stomatoyannopoulos, AW Nienhuis, P Leder, PW Majerus (eds.) Saunders, Philadelphia, 1987, pp 271–346

  7. Saier MH, Wu LF Jr, Reizer J: Regulation of bacterial physiological processes by three types of protein phosphorylating systems. TIBS 15: 391–395, 1990

    Google Scholar 

  8. Magnani M, Stocchi V, Serafini G, Chiarantini L, Fornaini G: Purification, properties and evidence for two subtypes of human placenta hexokinase type I. Arch Biochem Biophys 260: 388–399, 1988

    Google Scholar 

  9. Hunkapillar MV, Lujan E, Ostander F, Hood LE: Isolation of microgram quantities of proteins from polyacrylamide gels for amino acid sequence analysis. In: CHW Hirs, SN Timasheff (eds.) Methods in Enzymology. Academic Press, Orlando, vol 91, 1983, pp 227–236

    Google Scholar 

  10. Miller JM: Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, New York, 1972

    Google Scholar 

  11. Allen G: In: TS Work, RH Burdon (eds.) Sequencing of Proteins and Peptides: Laboratory techniques in Biochemistry and Molecular Biology. 1981, Elsevier/North Holland, New York

    Google Scholar 

  12. Chang JY: Manual micro-sequence analysis of polypeptides using dimethylaminoazobenzene isothiocyanate. In: CHW Hirs, SN Timasheff (eds.) Methods in Enzymology. Academic Press, New York, vol 91, 1983, pp 455–466

    Google Scholar 

  13. Chang JY, Brauer D, Wittmann-Liebold B: Micro-sequence analysis of peptides and proteins using 4-NN-dimethylaminoazobenzene 4′-isothiocyanate/phenylisothiocyanate double coupling method. FEBS Lett 93: 205–214, 1978

    Article  Google Scholar 

  14. Stocchi V, Piccoli G, Magnani M, Palma F, Biagiarelli B, Cucchiarini L: Reversed-phase high-performance liquid chromatography separation of dimethylaminoazobenzene thiohydantoin-amino acid derivatives for amino acid analysis and microsequencing studies at the picomole level. Anal Biochem: 107–117, 1989

  15. Chomczymski P, Sacchi N: Single-step method of RNA isolation by acid guanidium thiocynate-phenol-chloroform extraction. Anal Biochem 162: 156–159, 1987

    Article  CAS  PubMed  Google Scholar 

  16. Gubler U, Hoffman BJ: A simple and very efficient method for generation c-DNA libraries. Gene 25: 263–271, 1983

    Google Scholar 

  17. Young RA, Davis RW: Efficient isolation of genes by using antibody probes. Proc Natl Acad Sci USA 80: 1194–1198, 1983

    Google Scholar 

  18. Sanger F, Nicklen S, Culson AR: DNA sequencing with chain terminating inhibitors. Proc Natl Acad Sci USA 74: 5463–5467, 1977

    CAS  PubMed  Google Scholar 

  19. Lipman DJ, Pearson WR: Rapid and sensitive protein similarity searches. Science 227: 1435–1441, 1985

    Google Scholar 

  20. Thomas PS: Hybridization of denatured RNA transferred or dotted to nitrocellulose paper. In: R Wu, L Grossman, K Moldave (eds.) Methods in Enzymology. Academic Press, Orlando, vol 100, 1983, pp 255–266

    Google Scholar 

  21. Wang JYJ, Queen C, Baltimore D: Expression of an abelson murine leukemia virus-enclosed protein in Escherichia coli causes extensive phosphorylation of tyrosine residues. J Biol Chem 257: 13181–13184, 1982

    Google Scholar 

  22. Nishi S, Seino S, Bell GI: Human hexokinase: sequences of amino and carboxyl-terminal halves are homologous. Biochem Biophys Res Commun 157: 937–943, 1988

    Google Scholar 

  23. Schwab DA, Wilson JE: Complete amino acid sequence of rat brain hexokinase, deduced from the cloned cDNA, a proposed structure of a mammalian hexokinase. Proc Natl Acad Sci USA 86: 2563–2567, 1989

    Google Scholar 

  24. Arora KK, Fanciulli M, Pedersen PL: Glucose phosphorylation in tumor cells. Cloning, sequencing, and overexpression in active form of a full-length cDNA encoding a mitochondrial bindable form of hexokinase. J Biol Chem 265: 6481–6488, 1990

    Google Scholar 

  25. Wang JYJ, Koshland DE Jr: Regulatory protein phosphorylation in the prokariote Salmonella typhimurium. Cold String Harbor Cord Cell Proliferation 8: 637–643, 1981

    Google Scholar 

  26. Colowick SP: The Hexokinases. In: PD Boyer (ed.) The Enzymes. Academic Press, New York, 3rd ed, vol 9, 1973, pp 1–48

    Google Scholar 

  27. Schirch DM, Wilson JE: Rat brain hexokinase: amino acid sequence at the substrate hexose binding site is homologous to that of yeast hexokinase. Arch Biochem Biophys 257: 1–12, 1987

    Google Scholar 

  28. Nishizuka Y: The molecular heterogeneity of protein kinase C and its implications for cellular regulation, Nature 334: 661–665, 1988

    Google Scholar 

  29. Mozier NM, Zurcher-Neerly HA, Guido DM, Mathews WR, Heinrikson RL, Fraser ED, Walsh MP, Pearson JD: Amino acid sequence of a 12-KDa inhibitor of protein kinase C. Eur J Biochem 134: 19–23, 1990

    Google Scholar 

  30. Melloni E, Pontremoli S, Michetti M, Sacco O, Sparatore B, Horecker BL: The involvement of Calpain in the activation of Protein Kinase C in neutrophils by phorbol mizistic acid. J Biol Chem 261: 4101–4105, 1986

    Google Scholar 

  31. Huang FL, Glinsmann WL: Separation and characterization of two phosphorylase phosphatase inhibitor from rabbit skeletal muscle. Eur J Biochem 70: 419–426, 1976

    Google Scholar 

  32. Kyte J, Doolittle RF: A simple method for displaying the hydropathic character of a protein. J Mol Biol 157: 105–132, 1982

    CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Dipartimento di Genetica, Biologia e Chimica Medica, Università di Torino, Italy

    Aurora Daniele, Fiorella Altruda, Marina Ferrone & Lorenzo Silengo

  2. Istituto di Chimica Biologica ‘G. Fornaini’, Università degli Studi di Urbino, Italy

    Laura Chiarantini, Marzia Bianchi, Vilberto Stocchi & Mauro Magnani

Authors
  1. Aurora Daniele
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Fiorella Altruda
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Marina Ferrone
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Lorenzo Silengo
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Laura Chiarantini
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Marzia Bianchi
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Vilberto Stocchi
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Mauro Magnani
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Daniele, A., Altruda, F., Ferrone, M. et al. Cloning and expression of a new human polypeptide which regulates protein phosphorylation in Escherichia coli. Mol Cell Biochem 107, 87–94 (1991). https://doi.org/10.1007/BF00225511

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00225511

Key words

Search

Navigation