The small GTPase Rho is best known for its influence on cell shape, but a report in Developmental Cell by Jeffrey Settleman's group now shows that it might control cell size too.

In mammalian cells, a class of GTPase activating proteins — GAPs — are the main negative regulators of Rho, and p190-B RhoGAP seems to be among the most potent of these in vivo. To discover the biological function of p190-B RhoGAP, Settleman's group disrupted the gene in mice. The animals showed a range of defects and died immediately after birth. What was striking, though, was their 30% reduction in size and the intriguing similarity of their phenotype to that seen in mice that lack the transcription factor cyclic AMP responsive element binding protein (Creb). Further analysis showed that levels of phosphorylated Creb were reduced in the p190-B RhoGAP mutants, adding to the possibility of a connection between these two proteins.

The authors then compared cells from mutant and wild-type embryos and found that the mutant cells were 30% smaller, which probably accounted for the decrease in tissue size. Because cells that expressed a constitutively active form of Creb in p190-B RhoGAP−/− cells were larger than p190-B RhoGAP−/− parental cells, Creb probably functions downstream of p190-B RhoGAP. Furthermore, transfection of wild-type cells with constitutively actived Creb alone gave rise to abnormally large cells, whereas a dominant-negative Creb construct, or a constitutively actived form of Rho (RhoV14), which mimics the levels of active Rho that are seen in p190-B RhoGAP−/− cells, resulted in smaller cells.

Signalling by the insulin/insulin-growth factor (IGF) pathway is known to regulate the size of cells, so the group studied the effect of IGF on Creb phosphorylation in p190-B RhoGAP−/− cells and found it to be reduced compared with wild-type cells. The RhoV14 construct also had the same effect. One Rho effector — Rho-kinase (ROK) — is known to antagonize insulin signalling by associating with the insulin receptor substrate IRS, so could this account for the decrease in cell size and, if so, how? Adding a ROK inhibitor restored Creb phosphorylation and cell size in p190-B RhoGAP−/− cells, so ROK is indeed important in regulating cell size. And further analysis showed that phosphorylation of c-Jun-amino-terminal kinase (JNK) and p38 — two kinases that are downstream of insulin/IGF signalling — is reduced in p190-B RhoGAP−/− cells. As both these kinases activate Creb, this might well explain how Rho signalling affects cell size as well as its shape.