RAS proteins are important direct activators of p110α, p110γ, and p110δ type I phosphoinositide 3-kinases (PI3Ks), interacting via an amino-terminal RAS-binding domain (RBD). Here, we investigate the regulation of the ubiquitous p110β isoform of PI3K, implicated in G-protein-coupled receptor (GPCR) signaling, PTEN-loss-driven cancers, and thrombocyte function. Unexpectedly, RAS is unable to interact with p110β, but instead RAC1 and CDC42 from the RHO subfamily of small GTPases bind and activate p110β via its RBD. In fibroblasts, GPCRs couple to PI3K through Dock180/Elmo1-mediated RAC activation and subsequent interaction with p110β. Cells from mice carrying mutations in the p110β RBD show reduced PI3K activity and defective chemotaxis, and these mice are resistant to experimental lung fibrosis. These findings revise our understanding of the regulation of type I PI3K by showing that both RAS and RHO family GTPases directly regulate distinct ubiquitous PI3K isoforms and that RAC activates p110β downstream of GPCRs.

RAC1 P29 is the third most commonly mutated codon in human cutaneous melanoma, after BRAF V600 and NRAS Q61. Here, we study the role of RAC1^P29S in melanoma development and reveal that RAC1^P29S activates PAK, AKT, and a gene expression program initiated by the SRF/MRTF transcriptional pathway, which results in a melanocytic to mesenchymal phenotypic switch. Mice with ubiquitous expression of RAC1^P29S from the endogenous locus develop lymphoma. When expressed only in melanocytes, RAC1^P29S cooperates with oncogenic BRAF or with NF1-loss to promote tumorigenesis. RAC1^P29S also drives resistance to BRAF inhibitors, which is reversed by SRF/MRTF inhibitors. These findings establish RAC1^P29S as a promoter of melanoma initiation and mediator of therapy resistance, while identifying SRF/MRTF as a potential therapeutic target.

Volume depletion and/or increased hematocrit are associated with poor prognosis in necrotizing pancreatitis. Several studies suggest that intrathoracic blood volume index (ITBI) might be superior to central venous pressure (CVP) with regard to preload assessment. Therefore, the aim of our study was to evaluate the predictive value of CVP and hematocrit with regard to ITBI, and to correlate these parameters to cardiac index (CI). Prospective study. Medical intensive care unit, university hospital. Within 24 hrs of intensive care unit-admission, 96 hemodynamic measurements using the PiCCO system were performed in 24 patients with necrotizing pancreatitis. Mean CVP (12.11 ± 5.97 mm Hg; median 11.5 normal: 1–9 mm Hg) was elevated, whereas mean ITBI (822.8 ± 157.0 mL/m2; median 836 mL/m2; normal: 850–1000 mL/m2) was decreased. Fifty-one of 96 ITBI values were decreased (prevalence of hypovolemia of 53%). No CVP value was decreased. Fifty-three CVP measurements were elevated despite simultaneous ITBI levels indicating a normal or decreased preload. Sensitivity, specificity, positive predictive value, and negative predictive value of CVP with regard to volume depletion (ITBI 40% [female] or >44% [male]) was found in 11 of 51 measurements with decreased ITBI. Sensitivity, specificity, positive predictive value, and negative predictive value of an increase in hematocrit with regard to volume depletion according to ITBI were 22%, 82%, 58%, and 48%, respectively. ITBI and Δ-ITBI significantly correlated to CI and Δ-CI (r = .566, p < 0.001; r = .603, p < 0.001), respectively. CVP and Δ-CVP did not correlate to CI and Δ-CI, respectively. There was a significant correlation between ITBI and extravascular lung water index (r = .392; p < 0.001), but no correlation between CVP and extravascular lung water index (r = .074; p = 0.473). Volume depletion according to ITBI was found in more than half the patients. The predictive values of CVP and hematocrit with regard to volume depletion were low. ITBI and its changes significantly correlated to CI and its changes, which was not observed for CVP and Δ-CVP. Therefore, ITBI appears to be more appropriate for volume management in necrotizing pancreatitis than CVP or hematocrit.

The integrated stress response (ISR) integrates a broad range of environmental and endogenous stress signals to the phosphorylation of the α-subunit of eukaryotic translation initiation factor 2 (eIF2α). Although intense or prolonged activation of this pathway is known to induce apoptosis, the molecular mechanisms coupling stress-induced eIF2α phosphorylation to the cell death machinery have remained incompletely understood. In this study, we characterized apoptosis initiation in response to classical activators of the ISR (tunicamycin, UVC, elevated osmotic pressure, arsenite). We found that all applied stress stimuli activated a mitochondrial pathway of apoptosis initiation. Rapid and selective down-regulation of the anti-apoptotic BCL-2 family protein MCL-1 preceded the activation of BAX, BAK, and caspases. Stabilization of MCL-1 blocked apoptosis initiation, while cells with reduced MCL-1 protein content were strongly sensitized to stress-induced apoptosis. Stress-induced elimination of MCL-1 occurred with unchanged protein turnover and independently of MCL-1 mRNA levels. In contrast, stress-induced phosphorylation of eIF2α at Ser⁵¹ was both essential and sufficient for the down-regulation of MCL-1 protein in stressed cells. These findings indicate that stress-induced phosphorylation of eIF2α is directly coupled to mitochondrial apoptosis regulation via translational repression of MCL-1. Down-regulation of MCL-1 enables but not enforces apoptosis initiation in stressed cells.

Inhibitor of κB (IκB) β (IκBβ) represents one of the major primary regulators of NF-κB in mammals. In contrast to the defined regulatory interplay between NF-κB and IκBα, much less is known about the biological function of IκBβ. To elucidate the physiological role of IκBβ in NF-κB signaling in vivo, we generated IκBβ-deficient mice. These animals proved to be highly refractory to LPS-induced lethality, accompanied by a strong reduction in sepsis-associated cytokine production. In response to LPS, IκBβ is recruited to the IL-1β promoter forming a complex with the NF-κB subunits RelA/c-Rel required for IL-1β transcription. Further transcriptome analysis of LPS-stimulated wild-type and IκBβ-deficient BM-derived macrophages revealed several other genes with known regulatory functions in innate immunity arguing that a subset of NF-κB target genes is under control of IκBβ. Collectively, these findings provide an essential proinflammatory role for IκBβ in vivo, and establish a critical function for IκBβ as a transcriptional coactivator under inflammatory conditions.