Carlos Martínez-Campa

The hexokinase 2 protein participates in regulatory DNA‐protein complexes necessary for glucose repression of the SUC2 gene in Saccharomyces cerevisiae

The HXK2 gene plays an important role in glucose repression in the yeast Saccharomyces cerevisiae. Recently we have described that the HXK2 gene product, isoenzyme 2 of hexokinase, is located both in the nucleus and in the cytoplasm of S. cerevisiae cells. In this work we used deletion analysis to identify the essential part of the protein‐mediating nuclear localisation. Determinations of fructose‐kinase activity and immunoblot analysis using anti‐Hxk2 antibodies in isolated nuclei, together with observations of the fluorescence distribution of Hxk2‐GFP fusion protein in cells transformed with an HXK2::gfp mutant gene, indicated that the decapeptide KKPQARKGSM, located between amino acid residues 7 and 16 of hexokinase 2, is important for nuclear localisation of the protein. Further experimental evidence, measuring invertase activity in wild‐type and mutant cells expressing a truncated version of the Hxk2 protein unable to enter the nucleus, shows that a nuclear localisation of Hxk2 is necessary for glucose repression signalling of the SUC2 gene. Furthermore, we demonstrate using gel mobility shift analysis that Hxk2 participates in DNA‐protein complexes with cis‐acting regulatory elements of the SUC2 gene promoter.

Abnormal Properties of Mutants in the Hinge Region of ERα: Implications in Breast Cancer

In the search for differences between estrogen receptor (ER) α and ERβ, we proved that ERα but not ERβ directly interacts with calmodulin (CaM) through the hinge region. The transcriptional activity of a mutant unable to interact with CaM becomes insensitive to inhibition by CaM antagonists (W7). These residues are acetylated by p300 and substitution of lysine 302 and 303 with other residues enhance ERα-hormone sensitivity, suggesting that ERα acetylation normally suppresses ligand sensitivity. Also, the somatic mutation K303R has been identified in early premalignant breast lesions. ERα K303R normally binds E but shows increased E-induced transcriptional activation and increased proliferation in response to E when transfected into breast cancer cell (BC) lines. Herein, we show that mutations K303R and K303A, render an ERα unable to interact with CaM and therefore insensitive to W7. K303 homodimers and K303 mutant/wt heterodimers show increased sensitivity to E. Contrary to the wt ERα, AP1 transcriptional activity is inhibited by estradiol (E2) and OH-Tamoxifen (OH-TAM) in both K303R and K303A mutants.