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Xenopus oocytes were used as cellular and molecular sentinels to assess the effects of a new class of organometallic compounds called ferrocenyl dihydroquinolines that have been developed as potential anti-cancer agents. One ferrocenyl dihydroquinoline compound exerted deleterious effects on oocyte survival after 48 h of incubation at 100 μM. Two ferrocenyl dihydroquinoline compounds had an inhibitory effect on the resumption of progesterone induced oocyte meiosis, compared to controls without ferrocenyl groups. In these inhibited oocytes, no MPF (Cdk1/cyclin B) activity was detected by western blot analysis as shown by the lack of phosphorylation of histone H3. The dephosphorylation of the inhibitory Y15 residue of Cdk1 occurred but cyclin B was degraded. Moreover, two apoptotic death markers, the active caspase 3 and the phosphorylated histone H2, were detected. Only 7-chloro-1-ferrocenylmethyl-4-(phenylylimino)-1,4-dihydroquinoline ( 8 ) did not show any toxicity and allowed the assembly of a histologically normal metaphase II meiotic spindle while inhibiting the proliferation of cancer cell lines with a low IC 50 , suggesting that this compound appears suitable as an antimitotic agent.

To study protein function in cellular signaling, manual microinjection is a direct technique, but limited by the small size of many cells. The giant vertebrate cell, the Xenopus laevis oocyte, is a perfect model system to perform these studies. Oocytes are numerous and synchronous cells, arrested in the G2 phase of the cell cycle and easily amenable to biochemical, electrophysiological, and cytological studies. We describe how to microinject proteins or peptides in this model and we study, as an example, the Grb2 transduction cascade.

The injection of the Grb2 adapter in Xenopus oocytes promotes G2/M transition without stimulation from a receptor only the first day after the oocytes removal from the ovaries. This cell cycle reinitiation is Ras-dependent and requires the SH2 and SH3 domains of Grb2. The SH2 domain of Grb2 binds the tyrosine phosphorylated lipovitellin1, a homologue of the human apolipoprotein B. The N-SH3 domain of Grb2 is linked to a proline-rich sequence of the C2 domain of PLC-gamma1, PLC-gamma1 itself is linked, through its SH3 domain, to the C-terminal proline-rich region of Sos. When Grb2-PLC-gamma1-Sos is associated, PLC-gamma1 is not phosphorylated on Y783 but shows a phospholipase activity. Inhibition of lipovitellin 1 or PLC-gamma1 avoids Grb2-induced cell cycle reinitiation. Therefore, the Grb2-lipovitellin 1 association is the starting point of a novel signaling pathway, where PLC-gamma1 binds Grb2 and recruits Sos.

Grb14 belongs to the Grb7 family of adapter proteins and was identified as a negative regulator of insulin signal transduction. Its inhibitory effect on the insulin receptor kinase activity is controlled by a newly discovered domain called PIR. To investigate the biochemical and biophysical characteristics of this new domain, we cloned and purified recombinant PIR-SH2, PIR, and SH2 domains. The isolated PIR and PIR-SH2 domains were physiologically active and inhibited insulin-induced reinitiation of meiosis in the Xenopus oocytes system. However, NMR experiments on (15)N-labelled PIR revealed that it did not present secondary structure. These results suggest that the PIR domain belongs to the growing family of intrinsically unstructured proteins.

The role of Grb7 adapters, Grb7, Grb10, and Grb14, was investigated in Xenopus oocytes expressing fibroblast growth factor receptors (FGFR). FGF-induced maturation of FGFR-expressing oocytes was blocked by previous injection of Grb7 or Grb14, but not Grb10. This effect correlated with Grb7/14 binding to the receptor, and inhibition of the Ras-dependent pathway. Interestingly, the phosphorylated insulin receptor interacting region (PIR) and Src 2 homology domains (SH2) of Grb7 and Grb14 were differently implicated in the inhibition of FGFR signalling. This study provided further evidence for specificity of the biological action of the Grb7 adapters on receptor tyrosine kinase signalling.

Grb14 is a member of the Grb7 family of adapters and acts as a negative regulator of insulin-mediated signaling. Here we found that the protein kinase Czeta (PKCzeta) interacting protein, ZIP, interacted with Grb14. Coimmunoprecipitation experiments demonstrated that ZIP bound to both Grb14 and PKCzeta, thereby acting as a link in the assembly of a PKCzeta-ZIP-Grb14 heterotrimeric complex. Mapping studies indicated that ZIP interacted through its ZZ zinc finger domain with the phosphorylated insulin receptor interacting region (PIR) of Grb14. PKCzeta phosphorylated Grb14 under in vitro conditions and in CHO-IR cells as demonstrated by in vivo labeling experiments. Furthermore, Grb14 phosphorylation was increased under insulin stimulation, suggesting that the PKCzeta-ZIP-Grb14 complex is involved in insulin signaling. The PIR of Grb14, which also interacts with the catalytic domain of the insulin receptor (IR) and inhibits its activity, was preferentially phosphorylated by PKCzeta. Interestingly, the phosphorylation of Grb14 by PKCzeta increased its inhibitory effect on IR tyrosine kinase activity in vitro. The role of ZIP and Grb14 in insulin signaling was further investigated in vivo in Xenopus laevis oocytes. In this model, ZIP potentiated the inhibitory action of Grb14 on insulin-induced oocyte maturation. Importantly, this effect required the recruitment of PKCzeta and the phosphorylation of Grb14, providing in vivo evidences for a regulation of Grb14-inhibitory action by ZIP and PKCzeta. Together, these results suggest that Grb14, ZIP, and PKCzeta participate in a new feedback pathway of insulin signaling.