Metastasis: Wherefore Arf Thou?

SummaryThe small GTP-binding protein Arf6 is known to be an important regulator of the actin cytoskeleton and of cell motility associated with metastasis. A recent study identifies yet another role for Arf6 in metastasis — as a regulator of plasma-membrane-derived microvesicle release

Structure–activity relationship studies of QS11, a small molecule Wnt synergistic agonist

Both the Wnt/β-catenin signaling pathway and small GTPases of the ADP-ribosylation factors (ARF) family play important roles in regulating cell development, homeostasis and fate. The previous report of QS11, a small molecule Wnt synergist that binds to ARF GTPase-activating protein 1 (ARFGAP1), suggests a role for ARFGAP1 in the Wnt/β-catenin pathway. However, direct inhibition of enzymatic activity of ARFGAP1 by QS11 has not been established. Whether ARFGAP1 is the only target that contributes to QS11's Wnt synergy is also not clear. Here we present structure-activity relationship (SAR) studies of QS11 analogs in two assays: direct inhibition of enzymatic activity of purified ARFGAP1 protein and cellular activation of the Wnt/β-catenin pathway. The results confirm the direct inhibition of ARFGAP1 by QS11, and also suggest the presence of other potential cellular targets of QS11

Calmodulin protects cells from death under normal growth conditions and mitogenic starvation but plays a mediating role in cell death upon B-cell receptor stimulation

Calmodulin (CaM) is the main intracellular Ca(2+) sensor protein responsible for mediating Ca(2+) triggered processes. Chicken DT40 lymphoma B cells express CaM from the two genes, CaMI and CaMII. Here we report the phenotypes of DT40 cells with the CaMII gene knocked out. The disruption of the CaMII gene causes the intracellular CaM level to decrease by 60%. CaMII(−/−) cells grow more slowly and die more frequently as compared to wild type (wt) cells but do not exhibit significant differences in their cell cycle profile. Both phenotypes are more pronounced at reduced serum concentrations. Upon stimulation of the B-cell receptor (BCR), the resting Ca(2+) levels remain elevated after the initial transient in CaMII(−/−) cells. Despite higher Ca(2+) resting levels, the CaMII(−/−) cells are partially protected from BCR induced apoptosis indicating that CaM plays a dual role in apoptotic processes

GIT1 regulates synaptic structural plasticity underlying learning.

The signaling scaffold protein GIT1 is expressed widely throughout the brain, but its function in vivo remains elusive. Mice lacking GIT1 have been proposed as a model for attention deficit-hyperactivity disorder, due to alterations in basal locomotor activity as well as paradoxical locomotor suppression by the psychostimulant amphetamine. Since we had previously shown that GIT1-knockout mice have normal locomotor activity, here we examined GIT1-deficient mice for ADHD-like behavior in more detail, and find neither hyperactivity nor amphetamine-induced locomotor suppression. Instead, GIT1-deficient mice exhibit profound learning and memory defects and reduced synaptic structural plasticity, consistent with an intellectual disability phenotype. We conclude that loss of GIT1 alone is insufficient to drive a robust ADHD phenotype in distinct strains of mice. In contrast, multiple learning and memory defects have been observed here and in other studies using distinct GIT1-knockout lines, consistent with a predominant intellectual disability phenotype related to altered synaptic structural plasticity

Nuclear GIT2 is an ATM substrate and promotes DNA repair

Insults to nuclear DNA induce multiple response pathways to mitigate the deleterious effects of damage and mediate effective DNA repair. G-protein-coupled receptor kinase-interacting protein 2 (GIT2) regulates receptor internalization, focal adhesion dynamics, cell migration, and responses to oxidative stress. Here we demonstrate that GIT2 coordinates the levels of proteins in the DNA damage response (DDR). Cellular sensitivity to irradiation-induced DNA damage was highly associated with GIT2 expression levels. GIT2 is phosphorylated by ATM kinase and forms complexes with multiple DDR-associated factors in response to DNA damage. The targeting of GIT2 to DNA double-strand breaks was rapid and, in part, dependent upon the presence of H2AX, ATM, and MRE11 but was independent of MDC1 and RNF8. GIT2 likely promotes DNA repair through multiple mechanisms, including stabilization of BRCA1 in repair complexes; upregulation of repair proteins, including HMGN1 and RFC1; and regulation of poly(ADP-ribose) polymerase activity. Furthermore, GIT2-knockout mice demonstrated a greater susceptibility to DNA damage than their wild-type littermates. These results suggest that GIT2 plays an important role in MRE11/ATM/H2AX-mediated DNA damage responses

GIT1 KO mice show impairment in spatial memory in the Morris water maze.

<p>WT (n = 9, black symbols and bars) mice and GIT1 KO (n = 10, white symbols and bars) were trained over 6 days to find the hidden platform. Distance to find the platform (A and B), latency to find the platform (swim time, D and E), and swim speed (G and H) are shown. Swim distance and time were also analyzed for individual quadrants of the test pool (C and F). The average of two trials per day is plotted. All data is ± SEM. * <i>p</i><0.05; ** <i>p</i><0.01 in a two-way repeated measures ANOVA comparing KO to WT, followed by Holm-Sidak post-hoc within-trial comparison. φ <i>p</i><0.05; φφ <i>p</i><0.005 comparing daily performance of WT or KO to their own Day 1 performance. GIT1 KO mice traveled more distance and spent more time moving at a faster speed to find the hidden platform. GIT1 KO mice swam significantly less time and distance in the target quadrant, while swimming more time and distance in adjacent quadrants seeking escape.</p

Spontaneous and amphetamine-induced locomotor activity in GIT1 deficient mice on the F2 c57Bl/6J x 129Ola genetic background.

<p>A) GIT1 WT (n = 8, black circle) and GIT1-KO (n = 8, open square) mice on the c57 x Ola F2 genetic background were subjected to the open field test for 24 hours under the normal 12hr-12hr light-dark cycle. High overall activity at time points during the post-dark light phase are due to distinct high-activity outlier individuals at each instance. Total distance was summed for the dark (active) phase (B) and light (inactive) phase (C) following the dark phase. D) GIT1 WT (n = 8) and GIT1-KO (n = 8) mice were injected with 4 mg/kg amphetamine in their active (dark) phase. E) Total distance was summed for 1h and 2h following the drug injection. <i>p</i><0.001 in a two-way repeated measures ANOVA between genotypes over time; * <i>p</i><0.05, ** <i>p</i><0.01, *** <i>p</i><0.001 within time using a Holm-Sidak post-hoc test.</p