Skip to main content

Phosphopeptide mapping of proteins ectopically expressed in tissue culture cell lines


Post-translational modifications such as phosphorylation play a vital role in the regulation of protein function. In our study of the basic Helix-loop-Helix (bHLH) transcription factor HAND1, it was suspected that HAND1 was being phosphorylated during trophoblast giant cell differentiation and that coexpression of a constitutively active kinase with HAND1 resulted in changes in the proteins dimerization profile. In order to accurately document HAND1 phosphorylation and identify the resides being modified, we employed metabolic cell labeling with 32P of tissue culture cells coexpressing a Flag-epitope tagged HAND1 along with a number of active kinases and phosphatase subunits. We generated phosphopeptide maps of the phosphorylated HAND1 using the methods described below and linked these modifications to changes in HAND1 biological function.


  1. Firulli AB. A HANDful of questions: The molecular biology of the HAND-subclass of basic helix-loop-helix transcription factors. Gene 2003; 312C:27–40.

    Article  Google Scholar 

  2. Massari ME, Murre C. Helix-Loop-Helix Proteins: Regulators of Transcription in Eucaryotic Organisms. Molec Cell Biol 2000; 20(2):429–440.

    Article  PubMed  CAS  Google Scholar 

  3. Firulli BA, Howard MJ, McDaid JR, McIlreavey L, Dionne KM, Centonze VE, Cserjesi P, Virshup DM, Firulli AB. PKA, PKC and the Protein Phosphatase 2A Influence HAND factor dimerization: A Mechanisms for Tissue Specific Transcriptional Regulation. Mol Cell 2003; 12:1225–1237.

    Article  PubMed  CAS  Google Scholar 

  4. Firulli BA, Hadzic DB, McDaid JR, Firulli AB. The basic helix-loop-helix transcription factors dHAND and eHAND exhibit dimerization characteristics that suggest complex regulation of function. J Biol Chem 2000; 275(43):33567–33573.

    Article  PubMed  CAS  Google Scholar 

  5. Scott IC, Anson-Cartwright L, Riley P, Reda D, Cross JC. The HAND1 basic helix-loop-helix transcription factor regulates trophobblast differentiation via multiple mechanisms. Molec Cell Biol 2000; 20(2):530–541.

    Article  PubMed  CAS  Google Scholar 

  6. Castanon I, von Stetina S, Kass J, Baylies MK. Dimerization partners determine the activity of the Twist bHLH protein during Drosophila mesoderm development. Development 2001; 128:3145–3159.

    PubMed  CAS  Google Scholar 

  7. James G, Olson EN. Deletion of the regulatory domain of protein kinase C exposes regions in the hinge and catalytic domains that mediate nuclear targeting. J Cell Biol 1992; 116(4):863–874.

    Article  PubMed  CAS  Google Scholar 

  8. Firulli AB, Maibenco DC, Kinniburgh AJ. Triplex forming Ability of a c-myc Promoter element Predicts Promoter Strength. Arch Biochem Biophys 1994; 310(1):236–242.

    Article  PubMed  CAS  Google Scholar 

  9. Van Der Geer P, Luo K, Sefton BM, Hunter T. Phosphopeptide mapping and phosphoamino acid analysis on cellulose thin-layer-plates. 2nd ed. The practical approach series, ed. B.D. Hames. New York, NY: Oxford University Press; 1999, 97–126.

    Google Scholar 

Download references

Author information

Authors and Affiliations


Corresponding author

Correspondence to Anthony B. Firulli.

Additional information

Published: March 19, 2004.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Firulli, B.A., Virshup, D.M. & Firulli, A.B. Phosphopeptide mapping of proteins ectopically expressed in tissue culture cell lines. Biol. Proced. Online 6, 16–22 (2004).

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Issue Date:

  • DOI:

Indexing terms

  • Phosphorylation
  • Dimerization
  • Helix-Loop-Helix Motifs