Connexins: a myriad of functions extending beyond assembly of gap junction channels

Connexins constitute a large family of trans-membrane proteins that allow intercellular communication and the transfer of ions and small signaling molecules between cells. Recent studies have revealed complex translational and post-translational mechanisms that regulate connexin synthesis, maturation, membrane transport and degradation that in turn modulate gap junction intercellular communication. With the growing myriad of connexin interacting proteins, including cytoskeletal elements, junctional proteins, and enzymes, gap junctions are now perceived, not only as channels between neighboring cells, but as signaling complexes that regulate cell function and transformation. Connexins have also been shown to form functional hemichannels and have roles altogether independent of channel functions, where they exert their effects on proliferation and other aspects of life and death of the cell through mostly-undefined mechanisms. This review provides an updated overview of current knowledge of connexins and their interacting proteins, and it describes connexin modulation in disease and tumorigenesis

Characterization of a tumor-associated activating mutation of the p110β PI 3-kinase.

The PI3-kinase pathway is commonly activated in tumors, most often by loss of PTEN lipid phosphatase activity or the amplification or mutation of p110α. Oncogenic mutants have commonly been found in p110α, but rarely in any of the other catalytic subunits of class I PI3-kinases. We here characterize a p110β helical domain mutation, E633K, first identified in a Her2-positive breast cancer. The mutation increases basal p110β activity, but does not affect activation of p85/p110β dimers by phosphopeptides or Gβγ. Expression of the mutant causes increases in Akt and S6K1 activation, transformation, chemotaxis, proliferation and survival in low serum. E633 is conserved among class I PI3 Ks, and its mutation in p110β is also activating. Interestingly, the E633K mutant occurs near a region that interacts with membranes in activated PI 3-kinases, and its mutation abrogates the requirement for an intact Ras-binding domain in p110β-mediated transformation. We propose that the E633K mutant activates p110β by enhancing its basal association with membranes. This study presents the first analysis of an activating oncogenic mutation of p110β

Effect of p110β mutant on transformation and chemotaxis.

<p>(A) NIH 3T3 cells stably expressing wild type or E633K p110β were plated in soft agar and colonies were counted after 3 weeks. Colony counts are normalized to the number of colonies produced by cells expressing p110β alone. (B) Equal number of NIH 3T3 cells stably expressing wild type or E633K p110β were plated and left to grow to confluence for 10 days. Foci were counted and normalized to cells expressing wild-type p110β. (C) NIH 3T3 cells stably expressing wild type or E633K p110β were starved overnight and plated either in 0% or 10% NCS in transwell chambers, and incubated with media containing 0% or 10% NCS in the lower chamber and upper chambers as indicated. Data are mean ± SEM of triplicate samples from two experiments.</p

Akt signaling, proliferation and survival of cells expressing mutant p110β.

<p>(A) Expression level of wild-type or E633K myc-p110β in stably-transfected cells. (B) Cells stably expressing wild type or E633K p10β were incubated overnight in 10%, 0.5% or 0% NCS media. Whole cell lysates were analyzed by western blotting with anti-pT308 Akt, anti-pT389 S6K, and anti-β-actin antibodies. (C-E) Cells stably expressing wild-type or E633K p110β were plated in 96-well plates, incubated for 24 and 48 hours in (C) 10% NCS medium, (D) 0.5% NCS medium, or (E) 0% NCS medium, and assayed using the MTT assay. (F) Cells stably expressing wild type or E633K p110β were incubated for 24 hours in 10%, 0.5%, or 0% NCS medium. Cell viability was assayed by Trypan blue staining. Dead cells are displayed as percent of total number of cells. Data are mean ± SEM of triplicate samples from two separate experiments.</p

Role of kinase activity on the increased proliferation and migration by the p110β mutant.

<p>(A) Cells stably expressing wild-type or E633K p110β were plated in 96-well plates, incubated for 24 and 48 hours in 10% NCS, with or without 200 nM TGX-221, and assayed using the MTT assay. (B) NIH 3T3 cells stably expressing wild type or E633K p110β were starved overnight and plated either in 0% or 10% NCS in transwell chambers, and incubated with media containing 0% or 10% NCS in the lower chamber and upper chambers as indicated, with or without 200 nM TGX-221 in both chambers as indicated. Data are mean ± SEM of at least duplicate samples from two separate experiments.</p