The P34G mutation reduces the transforming activity of K-Ras and N-Ras in NIH 3T3 cells but not of H-Ras.

Ras proteins (H-, N-, and K-Ras) operate as molecular switches in signal transduction cascades controlling cell proliferation, differentiation, or apoptosis. The interaction of Ras with its effectors is mediated by the effector-binding loop, but different data about Ras location to plasma membrane subdomains and new roles for some docking/scaffold proteins point to signaling specificities of the different Ras proteins. To investigate the molecular mechanisms for these specificities, we compared an effector loop mutation (P34G) of three Ras isoforms (H-, N-, and K-Ras4B) for their biological and biochemical properties. Although this mutation diminished the capacity of Ras proteins to activate the Raf/ERK and the phosphatidylinositol 3-kinase/AKT pathways, the H-Ras V12G34 mutant retained the ability to cause morphological transformation of NIH 3T3 fibroblasts, whereas both the N-Ras V12G34 and the K-Ras4B V12G34 mutants were defective in this biological activity. On the other hand, although both the N-Ras V12G34 and the K-Ras4B V12G34 mutants failed to promote activation of the Ral-GDS/Ral A/PLD and the Ras/Rac pathways, the H-Ras V12G34 mutant retained the ability to activate these signaling pathways. Interestingly, the P34G mutation reduced specifically the N-Ras and K-Ras4B in vitro binding affinity to Ral-GDS, but not in the case of H-Ras. Thus, independently of Ras location to membrane subdomains, there are marked differences among Ras proteins in the sensitivity to an identical mutation (P34G) affecting the highly conserved effector-binding loop.

Polymorphisms G691S/S904S of RET as genetic modifiers of MEN 2A.

Multiple endocrine neoplasia type 2A (MEN 2A) is associated with specific germ-line missense mutations in the RET proto-oncogene. Only a minor fraction of human disorders are simple monogenic diseases, and the identification of polymorphisms that increase susceptibility, including variations in pathological phenotypes, to human diseases is one of the key problems in medical genetics. To explore this idea, we analyzed the polymorphisms G691S (exon 11) and S904S (TCC-TCG, exon 15) of RET in 198 individuals corresponding to 35 unrelated Spanish MEN 2A families (104 patients with oncogenic MEN 2A mutation and 94 healthy relatives). We found strong cosegregation between both polymorphisms (100% Fisher's exact test, P < 0.001) using a control population containing 653 healthy individuals (362 females and 291 males). Interestingly, we found that the homozygous for these polymorphisms were, on average, 10 years younger at diagnosis compared with heterozygous and wild-type homozygous (P = 0.037). Taken together, all these findings could indicate that the G691S and S904S variants of RET have a modifier effect on the age at onset of MEN 2A. Moreover, compared with the control population, the homozygote status was significantly more prevalent in a series of 110 sporadic thyroid carcinoma (odds ratio = 2.36), suggesting that these polymorphisms may play a role as a low penetrance risk factor.

HSos1 contains a new amino-terminal regulatory motif with specific binding affinity for its pleckstrin homology domain.

The protein hSos1 is a Ras guanine nucleotide exchange factor. In the present study, we investigated the function of the amino-terminal region of the hSos1 protein, corresponding to the first 600 residues, which includes the Dbl and pleckstrin homology (DH and PH) domains. We demonstrated, using a series of truncated mutants, that this region is absolutely necessary for hSos1 activity. Our results suggest that the first 200 residues (upstream of DH domain), which we called the HF motif on the basis of their homology with histone H2A, may exert negative control over the functional activity of the whole hSos1 protein. In vitro binding analysis showed that the HF motif is able to interact specifically with the PH domain of hSos1. The amino-terminal region of hSos1 may be associated in vivo with an expressed HF motif. These findings document the existence of the HF motif located upstream of the DH domain in the hSos1 protein. This motif may be responsible for the negative control of hSos1, probably by intramolecular binding with the PH domain.

Genetic analysis of RET, GFR alpha 1 and GDNF genes in Spanish families with multiple endocrine neoplasia type 2A.

Multiple endocrine neoplasia type 2A (MEN 2A) is associated with specific germline missense mutations in the RET proto-oncogene. This locus encodes a receptor tyrosine kinase whose activation requires the formation of a multimeric receptor complex including GDNF as a ligand and GFR alpha 1 as a coreceptor. In order to explore the role of RET, GFR alpha 1 and GDNF genes in the variation of phenotypes observed in MEN2A families, we analysed germline mutations of these genes in 4 unrelated Spanish MEN2A families (23 cases studied). We found 2 novel variants corresponding to a single change in position + 47 (intron 12) of RET and position +22 (intron 7) of GFR alpha 1. Furthermore, we observed strong co-segregation between 2 polymorphisms of RET [G691S (exon 11) and S904S (TCC-TCG, exon 15) (100%, Fisher's exact test, p< 0.001)]. More interestingly, we found that these polymorphisms occurred at a significantly high frequency in patients with age at onset < 20 years old (Kruskal-Wallis's and Fisher's exact test, p = 0.007). These findings suggest that the G691S and S904S variants of RET may somehow play a role on the age of onset of MEN 2A.

p53/MDM2 Pathway Aberrations in Parathyroid Tumors: p21(WAF-1) and MDM2 Are Frequently Overexpressed in Parathyroid Adenomas.

Parathyroid adenomas (PTAs) are the main cause of primary hyperparathyroidism. Cell cycle regulation in normal parathyroid tissue (NPT) and PTA remains largely unknown. We have systematically explored several components involved in the p53/MDM2/p19(ARF) pathway in PTA and compared the results were with NPT. Forty-six PTA and 12 NPT were immunostained with anti-p21(WAF-1), MDM2, p53, and p27(KP1) antibodies. The slides were processed by cytometry and the results were statistically analyzed using nonparametric methods (Mann-Whitney test). p2l(WAF-1) and MDM2 expression were significantly higher in PTA compared with NPT (p < 0.05). The opposite results were found for p27(KIP1) (p< 0.05). Occasional p53 staining was found in some PTA, albeit no significant difference was found in comparison with NPT. In conclusion, MDM2 and p2l(WAF-1) are the proteins more overexpressed in PTA. These findings are surprising taking into account the benign nature of PTA, making them suitable candidates for further molecular analysis.

Analysis of the Cycilin D1/p16/pRb Pathway in Parathyroid Adenomas.

Cyclin D1/p16/pRb pathway controlling G1-S cell cycle checkpoint is frequently altered in human tumors. Currently, scarce data are available for parathyroid tumors. We have studied 46 parathyroid adenomas (PTAs) and 12 normal parathyroid glands (PTGs) by immunohistochemistry with cyclin D1 (CD1), p16, pRb, and Ki-67 antibodies. We observed CD1 expression in 89%, p16 in 70%, and pRb in 100% of PTAs. Statistically significant differences with normal PTGs were found only concerning p16 expression (p <0.05). Proliferating rate (Ki-67) was always low, although significantly higher than in normal PTGs. Our findings demonstrate the presence of alterations in the CD1/p16/pRb pathway in PTAs, consisting in p16 overexpression apparently unrelated to pRb downregulation. On the other hand, we did not find significant differences in CD1 expression between PTAs and normal PTGs, suggesting CD1 overexpression could be a physiological event in parathyroid tissue.