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. Youseli, 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 approximately 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.