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Evidence for an important role of serine 16 and its phosphorylation in the stabilization of c-Mos

Oncogene volume 18, pages 4287–4294 (1999)Cite this article

Abstract

The c-Mos serine/threonine protein kinase is an essential component of cytostatic factor (CSF), which is required for metaphase II arrest of eggs in vertebrates. Previously, we showed that c-Mos residue Ser-16 is phosphorylated in the ts110 Mo-MuSV-encoded Gag-Mos fusion protein. Here we provide evidence that Mos is phosphorylated at Ser-16 in transfected COS-1 cells. To investigate the role of this phosphorylation, Ser-16 was substituted with alanine or glutamic acid in full-length v-Mos (an Env-Mos fusion protein that contains 31 additional amino acids at the amino terminus of c-Mos), its mouse c-Mos equivalent version (v-Mos residues 32-374, hereafter referred to as Mos), and mouse c-Mos. Constructs expressing mutant versions of Mos were transfected into COS-1 and NIH3T3 cells in a transient and stable manner, respectively. Synthesis and proteolysis of Mos were evaluated by pulse-chase analysis of 35S-methionine-labeled proteins. Our findings indicate that the S16A mutant of Mos was highly unstable. It accumulated to approximately 10% of the level of wild-type Mos or its S16E mutant. In addition, the S16A mutation but not the S16E mutation inhibited Mos interaction with a cellular protein, p35, suggesting that phosphorylation at Ser-16 may promote Mos interaction with p35. As expected from its destabilizing effect, the S16A mutation caused a dramatic decrease in the cellular transforming activity of Mos (determined by soft-agar colony-formation assay with the stably transfected NIH3T3 cells), which is known to correlate with its CSF function. Efficient ubiquitin-mediated proteolysis of c-Mos requires proline as the second residue from the amino-terminus. In contrast to Mos, neither the stability nor protein kinase activity of v-Mos (in which c-Mos residue Pro-2 becomes Pro-33) was affected by the S16A mutation. To provide further proof that, similar to c-Mos, the S16A mutant is recognized by the proteolysis system through Pro-2, we show that the effect of the S16A mutation is reversed by the Pro-2-Ala mutation. Thus, our results indicate that Ser-16 has an important role in the regulation of c-Mos and that phosphorylation at Ser-16 may inhibit proteolysis of c-Mos.

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Acknowledgements

We thank Ralph Arlinghaus for valuable suggestions and critical reading of the manuscript. We also thank Linda Kimbrough for typing and Lore Feldman for editing the manuscript. This work was supported by grants R01 CA45125 and CA16672 (core) from the National Institutes of Health.

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Author notes

  1. Chau D Pham and Vijayalakshmi B Vuyyuru: CD Pham and VB Vuyyuru contributed equally to this work

Authors and Affiliations

  1. Department of Molecular Pathology, Box 172, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030, Texas, USA

    Chau D Pham, Vijayalakshmi B Vuyyuru, Yandan Yang, Wenlong Bai & Balraj Singh

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Pham, C., Vuyyuru, V., Yang, Y. et al. Evidence for an important role of serine 16 and its phosphorylation in the stabilization of c-Mos. Oncogene 18, 4287–4294 (1999). https://doi.org/10.1038/sj.onc.1202804

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