Sik1 Controls Cardiomyogenesis number of beating colonies was significantly decreased in sik1flp/flp EBs and analysis of cardiac markers revealed a down-regulation of terminal cardiac markers (aMyHC, Mlc2v, aCach and cTnI). When analyzed in depth, these cells showed a significant delay in cardiac differentiation in the absence of sik1, due to a defect in cell cycle exit of differentiating cardiomyoblasts. Gene profiling studies were performed to identify genes that may play a role in regulating sik specific function. One of the most down-regulated genes with cardiac expression was cdkn1c codifying for p57Kip2, a cyclin- dependent kinase inhibitor (CDKI), which showed a peculiar transcriptional regulation during cardiac differentiation, lost in the absence of sik1. Forced expression of p57Kip2 in sik1-deficient ESCs was able to rescue the mutant phenotype. Our data thus indicate that sik1 via p57Kip2 might have a central role in the control of the exit of cardiomyoblasts from the cell cycle toward the terminal differentiation of cardiomyocytes. Results Generation of sik1flp/flp ES Cell Clone To study the role of the sik1 gene during cardiomyocyte differentiation, we used a gene-trap ES cell line (GC389) (http:// genetrap.tigem.it/public) [14] carrying a pFlipa1 vector insertion, as shown in figure 1. The fusion transcript generated by the gene- trap vector insertion directs the expression of a truncated protein carrying only the N-terminal domain of SIK1 (residues 1 to 249) fused to the b-geo cassette (Fig. 1A). We mutated the second allele of sik1 by cultivating heterozygous mutant cells (sik1wt/flp) in increasing concentrations of the neomycin analogue G418, as previously described [15]. Several independent surviving clones were isolated and analyzed by RT-PCR to discriminate cells that were homozygous for the trapped allele (sik1flp/flp) over sik1wt/flp cells. Clones carrying the gene trap insertion on both alleles that did not express the wild-type transcript anymore Figure 1. Effects of gene trapping insertion and screening of homozygous mutant ES cells. (A) Schematic representation of the sik1 gene encoding for a protein of 779 amino acids. The SIK1 protein structure is visualized: the kinase domain (27 to 278), the UBA motif (303–343), the PKA motif (567–584) and the nuclear localization signal (586 to 612). The insertion of the gene-trap vector occurred in the seventh intron of the wild-type sik1 gene, causing a deletion of the protein segment from residue 250 to 779. The corresponding fusion transcript and the resulting protein are depicted. (B) Identification of homozygous ES clones for the trapped allele with RT-PCR analysis. Schematic representation of the oligonucleotides used to discriminate between the wild-type and fusion transcripts. X, Y and Z correspond to primers sik1UP, sik1LW and L232, respectively. The amplification of wild-type and mutant cDNA results in two products of different molecular weight, 682 bp and 378 bp respectively. Lanes: 1,2 and 3, amplification of sik1wt/wt cDNA; 4,5 and 6, amplification of sik1wt/flp cDNA; 7,8 and 9 amplification of sik1flp/flp cDNA; 10, sik1wt/wt total RNA; 11 and 12, sik1wt/flp total RNA; 13, sik1flp/flp total RNA. The arrow indicates the 500-bp band of the DNA molecular weight marker. The arrow indicates the 500-bp band of the DNA molecular weight marker. (C) Western blot on proteins extracted from wild-type and sik1flp/flp ES cells using an antibody against the b-galactosidase. In sik1flp/flpES cells is possible to detect a band corresponding to the fusion protein that is absent in wild-type ES cells. Different amounts of proteins were used on western blot. (D) Expression plasmids (6 mg) for FLAG-tagged sik1 and its truncated version (1–249aa) were transfected into HEK293T cells and subjected to immunoprecipitation with FLAG-M2 resin. Aliquots of FLAG-SIK1 wt and truncated IPs were subjected to western blotting (WB) using an anti-FLAG antibody (left panel) and to in vitro kinase assays using [c-32P]- ATP with AMARA peptide as a substrate (right panel). The relative activation was calculated by subtracting counts incorporated in the assay of non-immune sample from gross counts of immunoprecipitated samples to determine net cpm. (E) Intracellular localization of HA-tagged wild-type and mutant SIK1 proteins in HEK293T cells. doi:10.1371/journal.pone.0009029.g001 PLoS ONE | www.plosone.org 2 February 2010 | Volume 5 | Issue 2 | e9029