Negative regulation of receptor tyrosine kinases: unexpected links to c-Cbl and receptor ubiquitylation.

Intracellular signals mediated by the family of receptor tyrosine kinases play pivotal roles in morphogenesis, cell fate determination and pathogenesis. Precise control of signal amplitude and duration is critical for the fidelity and robustness of these processes. Activation of receptor tyrosine kinases by their cognate growth factors not only leads to propagation of the signal through various biochemical cascades, but also sets in motion multiple attenuation mechanisms that ultimately terminate the active state. Early attenuators pre-exist prior to receptor activation and they act to limit signal propagation. Subsequently, late attenuators, such as Lrig and Sprouty, are transcriptionally induced and further act to dampen the signal. Central to the process of signaling attenuation is the role of the E3 ubiquitin ligase c-Cbl. While Cbl-mediated processes of receptor ubiquitylation and endocytosis are relatively well understood, the links of Cbl to other negative regulators are just now beginning to be appreciated. Here we review some emerging interfaces between Cbl and the transcriptionally induced negative regulators Lrig and Sprouty.

Epigen, the last ligand of ErbB receptors, reveals intricate relationships between affinity and mitogenicity.

Four ErbB receptors and multiple growth factors sharing an epidermal growth factor (EGF) motif underlie transmembrane signaling by the ErbB family in development and cancer. Unlike other ErbB proteins, ErbB-2 binds no known EGF-like ligand. To address the existence of a direct ligand for ErbB-2, we applied algorithms based on genomic and cDNA structures to search sequence data bases. These searches reidentified all known EGF-like growth factors including Epigen (EPG), the least characterized ligand, but failed to identify novel factors. The precursor of EPG is a widely expressed transmembrane glycoprotein that undergoes cleavage at two sites to release a soluble EGF-like domain. A recombinant EPG cannot stimulate cells singly expressing ErbB-2, but it acts as a mitogen for cells expressing ErbB-1 and co-expressing ErbB-2 in combination with the other ErbBs. Interestingly, soluble EPG is more mitogenic than EGF, although its binding affinity is 100-fold lower. Our results attribute the anomalous mitogenic power of EPG to evasion of receptor-mediated depletion of ligand molecules, as well as to inefficient receptor ubiquitylation and down-regulation. In conclusion, EPG might represent the last EGF-like growth factor and define a category of low affinity ligands, whose bioactivity differs from the more extensively studied high affinity ligands.

LRIG1 restricts growth factor signaling by enhancing receptor ubiquitylation and degradation.

Kekkon proteins negatively regulate the epidermal growth factor receptor (EGFR) during oogenesis in Drosophila. Their structural relative in mammals, LRIG1, is a transmembrane protein whose inactivation in rodents promotes skin hyperplasia, suggesting involvement in EGFR regulation. We report upregulation of LRIG1 transcript and protein upon EGF stimulation, and physical association of the encoded protein with the four EGFR orthologs of mammals. Upregulation of LRIG1 is followed by enhanced ubiquitylation and degradation of EGFR. The underlying mechanism involves recruitment of c-Cbl, an E3 ubiquitin ligase that simultaneously ubiquitylates EGFR and LRIG1 and sorts them for degradation. We conclude that LRIG1 evolved in mammals as a feedback negative attenuator of signaling by receptor tyrosine kinases.

Regulation of fish gonadotropins.

Neurohormones similar to those of mammals are carried in fish by hypothalamic nerve fibers to regulate directly follicle-stimulating hormone (FSH) and luteinizing hormone (LH). Gonadotropin-releasing hormone (GnRH) stimulates the secretion of FSH and LH and the expression of the glycoprotein hormone alpha (GPalpha), FSHbeta, and LHbeta, as well as their secretion. Its signal transduction leading to LH release is similar to that in mammals although the involvement of cyclic AMP-protein kinase A (cAMP-PKA) cannot be ruled out. Dopamine (DA) acting through DA D2 type receptors may inhibit LH release, but not that of FSH, at sites distal to activation of protein kinase C (PKC) and PKA. GnRH increases the steady-state levels of GPalpha, LHbeta, and FSHbeta mRNAs. Pituitary adenylate cyclase-activating polypeptide (PACAP) 38 and neuropeptide Y (NPY) potentiate GnRH effect on gonadotropic cells, and also act directly on the pituitary cells. Whereas PACAP increases all three subunit mRNAs, NPY has no effect on that of FSHbeta. The effect of these peptides on the expression of the gonadotropin subunit genes is transduced differentially; GnRH regulates GPalpha and LHbeta via PKC-ERK and PKA-ERK cascades, while affecting the FSHbeta transcript through a PKA-dependent but ERK-independent cascade. The signals of both NPY and PACAP are transduced via PKC and PKA, each converging at the ERK level. NPY regulates only GPalpha- and LHbeta-subunit genes whereas PACAP regulates the FSHbeta subunit as well. Like those of the mammalian counterparts, the coho salmon LHbeta gene promoter is driven by a strong proximal tripartite element to which three different transcription factors bind. These include Sf-1 and Pitx-1 as in mammals, but the function of the Egr-1 appears to have been replaced by the estrogen receptor (ER). The GnRH responsive region in tilapia FSHbeta 5' flanking region spans the canonical AP1 and CRE motifs implicating both elements in conferring GnRH responsiveness. Generally, high levels of gonadal steroids are associated with high LHbeta transcript levels whereas those of FSHbeta are reduced when pituitary cells are exposed to high steroid levels. Gonadal or hypophyseal activin also participate in the regulation of FSHbeta and LHbeta mRNA levels. However, gonadal effects are dependent on the gender and stage of maturity of the fish.

Src promotes destruction of c-Cbl: implications for oncogenic synergy between Src and growth factor receptors.

Cellular Src and epidermal growth factor receptor (EGFR) collaborate in the progression of certain human malignancies, and their cooverexpression characterizes relatively aggressive animal tumors. Our study addressed the mode of oncogenic cooperation and reports that overexpression of c-Src in model cellular systems results in the accumulation of EGFR at the cell surface. The underlying mechanism involves inhibition of the normal, c-Cbl-regulated process of ligand-induced receptor down-regulation. In response to activation of c-Src, c-Cbl proteins undergo tyrosine phosphorylation that promotes their ubiquitylation and proteasomal destruction. Consequently, ubiquitylation of EGFR by c-Cbl is restrained in Src-transformed cells, and receptor sorting to endocytosis is impaired. In conclusion, by promoting destruction of c-Cbl, c-Src enables EGFR to evade desensitization, which explains Src-EGFR collaboration in oncogenesis.

Pituitary adenylate cyclase activating polypeptide and neuropeptide Y regulation of gonadotropin subunit gene expression in tilapia: role of PKC, PKA and ERK.

There is ample information on the hypophysiotropic function of pituitary adenylate cyclase-activating polypeptide (PACAP) and neuropeptide Y (NPY) in fish as in mammals, although evidence as to their direct effects on gonadotropic cells is scarce. We have previously reported that NPY and PACAP38 augment gonadotropin-releasing hormone (GnRH)-induced expression of glycoprotein alpha (alpha) subunit gene in the teleost fish, tilapia. The aim of the present study was to elucidate possible direct effects of these peptides on gonadotropin subunit gene expression in culture of tilapia pituitary cells, as well as the transduction pathways involved. Both NPY and PACAP38 (0.001-10 nM) increased the level of phosphorylated extracellular signal-regulated kinase (pERK) dose-dependently, reaching a peak at 0.1 and 0.01 nM, respectively. Inhibition of protein kinase C (PKC) by GF109203X (GF; 0.01-10 nM) suppressed NPY-stimulated pERK levels and its effect on alpha and luteinizing hormone (LH) beta subunit mRNA levels. However, NPY had no effect on follicle stimulating hormone (FSH) beta mRNA levels. NPY-elevated alpha, LHbeta mRNA and pERK levels were also attenuated by inhibition of protein kinase A (PKA) with H89 (0.01-10 nM). Exposure of the cells to the MAPK kinase (MEK) inhibitor (PD98059; PD 10, 25 and 50 microM) completely blocked NPY-induced ERK activity. In addition, this inhibitor abated the alpha and LHbeta mRNA responses to NPY. Similar experiments conducted to elucidate PACAP38 signaling revealed that PACAP38 (0.01 nM) elevated all three-gonadotropin subunit gene expression via both PKC-ERK and PKA-ERK cascades. It is suggested that both NPY and PACAP38 act directly on gonadotropes to elevate gonadotropin subunit gene expression. Whereas the expression of alpha and LHbeta subunit genes is regulated by both NPY and PACAP, the effect on the FSHbeta transcript is elicited only by PACAP38. NPY and PACAP38 stimulatory actions are mediated via protein kinase C (PKC) and protein kinase A (PKA), converging at the MEK-ERK cascade. These findings represent one of the fine tuning levels that differentially regulates gonadotropin subunit gene expression.