Drosophila C-Terminal Src Kinase Negatively Regulates Organ Growth and Cell Proliferation through Inhibition of the Src, Jun N-Terminal Kinase, and STAT Pathways

Normal development requires strict spatial and temporal control of cellular processes such as proliferation and differentiation for properly sized and functioning organisms to form. This control is achieved through a network of signal transduction pathways that coordinate developmental events between cells, tissues, and organs. Inappropriate activation of these signaling networks can cause diseases such as oncogenesis in which individual cells respond to aberrant internal cues to overproliferate. Src family cytoplasmic tyrosine kinases (SFKs) play important roles within these networks to regulate both developmental events and disease states, but the exact roles of SFKs in these events remain ambiguous. Humans and mice have at least eight SFKs, including Src, Fyn, and Yes. SFKs are composed of a tyrosine kinase domain, an SH2 domain, an SH3 domain, and a regulatory C-terminal region. They can be activated by receptor tyrosine kinases, cytokine receptors, G protein-coupled receptors, and integrins (reviewed in reference 70). SFK activation can cause cell cycle entry, cytoskeletal rearrangements, and alterations in cell adhesion (reviewed in reference 1). Mammalian tissue culture models have identified numerous downstream effectors of SFK functions; these include signaling molecules in the Ras/extracellular signal-regulated kinase (ERK), Jun kinase, Jak/STAT, and Rac/Rho pathways (70). SFK activities have not been well explored in vivo, however, in part due to functional redundancy among SFKs. For example, src−/− mice show only subtle osteoclast defects while src−/−; fyn−/−; yes−/− mouse embryos show early lethality and multiple developmental anomalies including neural tube defects and dramatically reduced size (37, 43, 63). Fibroblasts derived from src−/−; fyn−/−; yes−/− mice show reduced proliferation, suggesting that some of the phenotypes of compound knockouts are caused by proliferative defects during development (37). However, the precise roles of Src, Fyn, and Yes in the cell cycle during development remain unknown. SFKs are maintained in an inactive state through tyrosine phosphorylation of their C-terminal region by the negative regulator C-terminal Src kinase (Csk), which itself is closely related to SFKs (reviewed in reference 6). Deletion or mutation of the Csk target site leads to up-regulation of SFK activity (38, 52). Mammals have two Csk family members, Csk and Chk. Mice deficient for Csk show hyperactivation of SFKs and a striking embryonic phenotype also characterized by early lethality, neural tube defects, and reduced size (30, 49). Surprisingly, csk−/− fibroblasts do not show increased proliferation, which conflicts with data indicating that increased SFK activity leads to cell cycle entry. This could reflect functional compensation by Chk, which also negatively regulates SFKs (26, 58). The redundancy between multiple SFKs and Csk as well as the early lethality of Csk and compound SFK knockouts has impeded detailed evaluation of SFK function in developing tissues. Abnormal constitutive activation of SFKs has been implicated in oncogenesis (reviewed in reference 32). Numerous human tumors possess activated SFKs, but SFK mutations have been found in only a fraction of tumors. Some human colon cancers harbor mutations that abolish the ability of the C-terminal domain to inhibit Src kinase activity (31). The transforming v-Src oncogene shows deletion of the Csk target site (17). Since SFKs can be abnormally activated through disregulation of the C-terminal region, reduced Csk family kinase activity could promote oncogenesis. Yet, the role of Csk and/or Chk in tumors is controversial or unclear. Large deletions within the region of chromosome 15 that harbors Csk have been observed in colon cancers, the tumor types that commonly show elevated SFK activity (2, 7, 14, 19, 56), but no specific loss-of-function Csk mutations have been found in tumors to date. Reduced Csk expression and function is correlated with Src activation in primary hepatocellular tumors, primary colorectal tumors, and colon carcinoma cell lines (12, 45). However, others have reported elevated Csk in tumors with high SFK activity (73). In addition, csk−/− primary mouse fibroblasts do not show a transformed phenotype (30, 49). Perhaps mutations in other loci, such as chk, are required to reveal a tumor suppressor function for Csk (26, 58). A detailed exploration of Csk function in vivo is required to understand its role in disease, but again, such studies have been impeded by the early lethality of csk−/−mice. The imaginal disks of Drosophila melanogaster provide a powerful model system for the study of signal transduction. Imaginal discs share several properties with mammalian epithelial tissues: both are composed of epithelial cells that must maintain proportional growth, differentiation, and renewal to form functional tissues and organs. Cells within imaginal discs undergo proliferation and differentiation in response to molecular pathways that have been highly conserved across species and that function in oncogenesis. For example, studies of the eye imaginal disc have provided important evidence that the Ras and Jak/STAT signal transduction pathways are crucial for normal growth, proliferation, and differentiation (5, 25, 35, 44, 62). Recent genetic analyses of Drosophila tumor suppressor mutations have led to new insights about known human tumor suppressors and identification of new putative human tumor suppressors such as lats and salvador (34, 53, 67, 74). The Drosophila genome contains two SFKs, Src42A and Src64B, that are functionally similar to their mammalian counterparts (20, 66). Src42A and Src64B loss-of-function mutations disrupt cytoskeletal regulation within developing oocytes and embryos (20, 68). Yet the full repertoire of SFK functions remains to be elucidated in Drosophila. Src42A and Src64B are regulated by a Csk-like activity in flies, but until now, the gene responsible for that activity was unknown (42). In this report, we present the cloning and characterization of the Drosophila Csk ortholog, dCsk. Loss of dCsk function led primarily to overgrowth phenotypes in developing tissues such as the eye; genetic data indicated that excess proliferation was due to up-regulation of SFKs. We provide evidence that this overgrowth required the JNK and STAT signal transduction pathways. Reducing STAT function prevented growth and normal differentiation of dCsk mutant tissue, instead provoking dCsk−/− cells to undergo apoptosis. Our data provide in vivo evidence for a Src-dependent proapoptotic pathway triggered by reduced STAT function. They are consistent with results from Stewart et al. (64), published during submission of the manuscript of this report. Together, these results connect SFK signaling to the cell cycle and suggest an approach for restraining its proliferative potential.