Cellular and Molecular Neurobiology

, Volume 11, Issue 4, pp 415–433 | Cite as

Relationship among methylation, isoprenylation, and GTP binding in 21-to 23-kDa proteins of neuroblastoma

  • Roni Haklai
  • Yoel Kloog
Article

Summary

  1. 1.

    Dimethylsulfoxide-induced differentiated neuroblastoma express high levels of membrane 21 to 23-kDa carboxyl methylated proteins. Relationships among methylation, isoprenylation, and GTP binding in these proteins were investigated. Protein carboxyl methylation, protein isoprenylation, and [α-32P]GTP binding were determined in the electrophoretically separated proteins of cells labeled with the methylation precursor [methyl-3H]methionine or with an isoprenoid precursor [3H]mevalonate.

     
  2. 2.

    A broad band of GTP-binding proteins, which overlaps with the methylated 21 to 23-kDa proteins, was detected in [α-32P]GTP blot overlay assays. This band of proteins was separated in two-dimensional gels into nine methylated proteins, of which four bound GTP.

     
  3. 3.

    The carboxyl-methylated 21 to 23-kDa proteins incorporated [3H]mevalonate metabolites with characteristics of protein isoprenylation. The label was not removed by organic solvents or destroyed by hydroxylamine. Incorporation of radioactivity from [3H]mevalonate was enhanced when endogenous levels of mevalonate were reduced by lovastatin, an inhibitor of mevalonate synthesis. Lovastatin blocked methylation of the 21 to 23-kDa proteins as well (>70%).

     
  4. 4.

    Methylthioadenosine, a methylation inhibitor, inhibited methylation of these proteins (>80%) but did not affect their labeling by [3H]mevalonate. The results suggest that methylation of the 21 to 23-kDa proteins depends on, and is subsequent to, isoprenylation. The sequence of events may be similar to that known in ras proteins, i.e., carboxyl methylation of a C-terminal cysteine that is isoprenylated.

     
  5. 5.

    Lovastatin reduced the level of small GTP-binding proteins in the membranes and increased GTP binding in the cytosol. Methylthioadensoine blocked methylation without affecting GTP binding.

     
  6. 6.

    Thus, isoprenylation appears to precede methylation and to be important for membrane association, while methylation is not required for GTP binding or membrane association. The role of methylation remains to be determined but might be related to specific interactions of the small GTP-binding proteins with other proteins.

     

Key words

neuroblastoma carboxyl methylation isoprenylation G-proteins posttranslational modification 

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Copyright information

© Plenum Publishing Corporation 1991

Authors and Affiliations

  • Roni Haklai
    • 1
  • Yoel Kloog
    • 1
  1. 1.Department of BiochemistryThe George S. Wise Faculty of Life Sciences, Tel-Aviv UniversityTel-AvivIsrael

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