Constitutive Expression of Murine Sak-a Suppresses Cell Growth and Induces Multinucleation

Abstract

The murine Sak gene encodes two isoforms of a putative serine/threonine kinase, Sak-a and Sak-b, with a common N-terminal kinase domain and different C-terminal sequences. Sak is expressed primarily at sites where cell division is most active in adult and embryonic tissues (C. Fode, B. Motro, S. Yousefi, M. Heffernan, and J. W. Dennis, Proc. Natl. Acad. Sci. USA 91:6388–6392, 1994). In this study, we found that Sak-a transcripts were absent in growth-arrested NIH 3T3 cells, while in cycling cells, mRNA levels increased late in G₁ phase and remained elevated through S phase and mitosis before declining early in G₁. The half-life of hemagglutinin epitope-tagged Sak-a protein was determined to be ~2 to 3 h, and the protein was observed to be multiubiquitinated, a signal for rapid protein degradation. Overexpression of Sak-a protein inhibited colony-forming efficiency in CHO cells. Neither the Sak-b isoform nor Sak-a with a mutation in a strictly conserved residue in the kinase domain (Asp-154→Asn) conferred growth inhibition, suggesting that both the kinase domain and the C-terminal portion of Sak-a are functional regions of the protein. Sak-a overexpression did not induce a block in the cell cycle. However, expression of HA-Sak-a, but not HA-Sak-b, from a constitutive promoter for 48 h in CHO cells increased the incidence of multinucleated cells. Our results show that Sak-a transcript levels are controlled in a cell cycle-dependent manner and that this precise regulation is necessary for cell growth and the maintenance of nuclear integrity during cell division.