, a single dCbl
gene encodes two forms of dCbl proteins, which evolved into three homologues, c-Cbl, Cbl-b, and Cbl-c, in mammals (60
). As multidomain adaptor proteins with intrinsic E3 ubiquitin ligase activities, c-Cbl and Cbl-b have been implicated in diverse physiological processes, displaying characteristics of functional redundancy under certain circumstances. However, our understanding of the detailed mechanisms that underlie the physiological actions of dCbl has been limited, largely due to the myriad of signaling pathways that are subject to the positive or negative influences of Cbl proteins. In the present study, utilization of the genetic models of Drosophila
enabled us to examine the molecular evolution of Cbl actions. Our findings revealed the functional importance of dCbl in the IPCs in the brains of flies. dCbl cell autonomously inhibits the production of dILPs in IPCs and comprises an additional layer of coordination in insulin/IGF regulation of growth, carbohydrate metabolism, stress resistance, and longevity.
We found that dCbl in the IPCs of Drosophila
controls the biosynthesis of dILPs. dILPs are the ligands of the insulin/IGF-like signaling pathway that regulates growth, development, metabolism, and life span (4
). We demonstrated that disruption of dCbl in neurons or IPCs resulted in upregulation of dILPs, increased body growth, higher sensitivity to oxidative stress or starvation, decreased levels of trehalose, and shortened life span. The longevity and metabolic phenotypes can be explained by increased dILP production and signaling. We also observed that c-Cbl, a mammalian homologue, similarly inhibited insulin production and secretion in rat pancreatic β cells. However, whole-body deletion of c-Cbl does not affect the blood insulin levels in mice (60
), and abrogation of Cbl-b leads to an increase in insulin levels presumably due to insulin resistance resulting from adipose inflammation (18
). Thus, both c-Cbl and Cbl-b are likely to suppress insulin expression and secretion, and deficiency of one form may be functionally compensated for by the other form in mice. Conditional gene-targeting studies in mouse models are needed to clarify this issue.
It has been well established that Cbl downregulates the EGFR signaling pathway via its E3 ubiquitin ligase activity in both insects and mammals (51
). EGFR signaling has been documented to play a crucial role in postnatal growth (34
) as well as the expansion of pancreatic β-cell mass in response to feeding of a high-fat diet or during pregnancy (16
). The Cbl family members likely negatively regulate insulin biosynthesis and secretion in both insects and mammals by downregulating the EGFR/ERK pathway via their E3 ubiquitin ligase activity. Cbl may also regulate the growth and proliferation of β cells by downregulating other RTK pathways, such as the platelet-derived growth factor (PDGF) receptor signaling pathway (7
). PDGF signaling has been reported to be influenced by c-Cbl (35
). Additional mechanisms may also contribute to the observed phenotypes of flies with dCbl deficiency in neurons or IPCs as well as the enhancement of insulin secretion in INS-1 cells.
The molecular mechanisms by which dilp
genes are transcriptionally controlled are poorly understood in Drosophila
. Whereas neuronal or IPC-specific dCbl
suppression affected the expression of all three dilp
genes examined, EGFR signaling was genetically coupled to the regulation of dilp2
but not dilp5
. This further supports the notion that multiple signaling mechanisms may be involved in mediating dCbl regulation of dILP production. On an interesting note, distinct patterns of dilp
gene expression have also been reported in a recent study that implicated dERK signaling in mediating SNF (short neuropeptide F) regulation of dilp
expression within IPCs of Drosophila
). In mammalian β cells, a number of transcription factors, such as PDX-1, MafA, and NFAT, act to regulate insulin expression in response to ERK activation (21
). While we observed a c-Cbl deficiency-elicited augmentation of PDX-1 binding to the insulin promoter in INS-1 cells, it remains unclear whether other factors are also implicated. In addition, it has yet to be defined whether transcription factors in the dEGFR pathway, e.g., PntP1 (13
), or a PDX-1 analogue can act to mediate dCbl suppression of dILP production in Drosophila
. In this context, the molecular evolution of the regulatory factors involved in the transcriptional control of insulin/IGF1 represents an intriguing question that warrants more detailed investigations.
Recent studies suggest a common ancestral origin for both mammalian hypothalamic neurosecretory cells and the IPCs of flies (61
). Global c-Cbl-knockout mice (37
) and heterozygous mice expressing a C379A knock-in mutation within the RING finger domain of c-Cbl (38
) all had higher energy expenditure and improved peripheral insulin sensitivity. Given the central importance of the hypothalamic circuitries in the control of whole-body energy balance (22
), it is tempting to speculate that c-Cbl or Cbl-b may exert metabolic effects through modulating the hypothalamic neuroendocrine networks. Besides EGFR, neuronal Cbl may also target other signaling molecules, such as Src homology 2B (SH2B), that may interact with Cbl. We and others have previously shown that dSH2B, an adaptor protein that positively regulates insulin/IGF1 and leptin signaling pathways (40
), plays crucial roles in metabolism, stress resistance, and longevity in flies (54
). SH2B1, an SH2B family member, is required for the maintenance of normal body weight and glucose homeostasis in both humans and mice (2
). It is yet to be deciphered whether dCbl exerts its actions upon metabolism, stress tolerance, and life span through interacting with dSH2B to regulate the insulin/IGF1 pathway.
In summary, we show that knockdown of dCbl in neurons and IPCs results in increased dILP production and/or signaling activation, leading to reduced life span and stress tolerance. dCbl suppresses dILP production, at least in part, by downregulating the EGFR/ERK pathway through its E3 ligase activity. Moreover, Cbl suppression of insulin biosynthesis is evolutionarily conserved in mammalian β cells, suggesting that the Cbl pathway is critical for insulin production and secretion across animal species.