Hesso Farhan

MAPK signaling to the early secretory pathway revealed by kinase/phosphatase functional screening

An RNAi screen determines that the early secretory pathway is subject to phosphoregulation via a variety of signaling pathways, including a link between growth factor signaling and ER export.

The Golgi in cell migration : regulation by signal transduction and its implications for cancer cell metastasis

Migration and invasion are fundamental features of metastatic cancer cells. The Golgi apparatus, an organelle involved in posttranslational modification and sorting of proteins, is widely accepted to regulate directional cell migration. In addition, mounting evidence suggests that the Golgi is a hub for different signaling pathways. In this paper we will give an overview on how polarized secretion and microtubule nucleation at the Golgi regulate directional cell migration. We will review different signaling pathways that signal to and from the Golgi. Finally, we will discuss how these signaling pathways regulate the role of the Golgi in cell migration and invasion. We propose that by identifying regulators of the Golgi, we might be able to uncover unappreciated modulators of cell migration. Uncovering the regulatory network that orchestrates cell migration is of fundamental importance for the development of new therapeutic strategies against cancer cell metastasis.

Adaptation of endoplasmic reticulum exit sites to acute and chronic increases in cargo load

The biogenesis of endoplasmic reticulum (ER) exit sites (ERES) involves the formation of phosphatidylinositol‐4 phosphate (PI4) and Sec16, but it is entirely unknown how ERES adapt to variations in cargo load. Here, we studied acute and chronic adaptive responses of ERES to an increase in cargo load for ER export. The acute response (within minutes) to increased cargo load stimulated ERES fusion events, leading to larger but less ERES. Silencing either PI4‐kinase IIIα (PI4K‐IIIα) or Sec16 inhibited the acute response. Overexpression of secretory cargo for 24 h induced the unfolded protein response (UPR), upregulated COPII, and the cells formed more ERES. This chronic response was insensitive to silencing PI4K‐IIIα, but was abrogated by silencing Sec16. The UPR was required as the chronic response was absent in cells lacking inositol‐requiring protein 1. Mathematical model simulations further support the notion that increasing ERES number together with COPII levels is an efficient way to enhance the secretory flux. These results indicate that chronic and acute increases in cargo load are handled differentially by ERES and are regulated by different factors.

Parallel exploitation of diverse host nutrients enhances Salmonella virulence.

Pathogen access to host nutrients in infected tissues is fundamental for pathogen growth and virulence, disease progression, and infection control. However, our understanding of this crucial process is still rather limited because of experimental and conceptual challenges. Here, we used proteomics, microbial genetics, competitive infections, and computational approaches to obtain a comprehensive overview of Salmonella nutrition and growth in a mouse typhoid fever model. The data revealed that Salmonella accessed an unexpectedly diverse set of at least 31 different host nutrients in infected tissues but the individual nutrients were available in only scarce amounts. Salmonella adapted to this situation by expressing versatile catabolic pathways to simultaneously exploit multiple host nutrients. A genome-scale computational model of Salmonella in vivo metabolism based on these data was fully consistent with independent large-scale experimental data on Salmonella enzyme quantities, and correctly predicted 92% of 738 reported experimental mutant virulence phenotypes, suggesting that our analysis provided a comprehensive overview of host nutrient supply, Salmonella metabolism, and Salmonella growth during infection. Comparison of metabolic networks of other pathogens suggested that complex host/pathogen nutritional interfaces are a common feature underlying many infectious diseases.

Concentrative Export from the Endoplasmic Reticulum of the γ-Aminobutyric Acid Transporter 1 Requires Binding to SEC24D

Re-uptake of γ-aminobutyric acid (GABA) into presynaptic specializations is mediated by the GABA transporter 1 (GAT1), a member of the SLC6 gene family. Here, we show that a motif in the COOH terminus of GAT1 (566RL567), which is conserved in SLC6 family members, is a binding site for the COPII coat component Sec24D. We also identified residues in Sec24D (733DD734) that are required to support the interaction with GAT1 and two additional family members, i.e. the transporters for serotonin and dopamine. We used three strategies to prevent recruitment of Sec24D to GAT1: knock-down of Sec24D by RNA interference, overexpression of Sec24D-VN (replacement of 733DD734 by 733VN734), and mutation of 566RL567 to 566AS567 (GAT1-RL/AS). In each instance, endoplasmic reticulum (ER) export of GAT1 was impaired: in the absence of Sec24D or upon coexpression of dominant negative Sec24D-VN, GAT1 failed to undergo concentrative ER export; GAT1-RL/AS also accumulated in the ER and exerted a dominant negative effect on cell surface targeting of wild type GAT1. Our observations show that concentrative ER-export is contingent on a direct interaction of GAT1 with Sec24D; this also provides a mechanistic explanation for the finding that oligomeric assembly of transporters is required for their ER export: transporter oligomerization supports efficient recruitment of COPII components.

Signalling to and from the secretory pathway

For growth, survival, communication and homeostasis, cells transport a large number of proteins to the plasma membrane and the extracellular medium by using the secretory pathway. Consequently, to adapt to the surrounding environment and the different intracellular contexts, the secretory pathway needs to accommodate and respond to a plethora of endogenous and exogenous stimuli. It is now well established that several kinases, known to be activated by environmental stimuli, signal from the plasma membrane to the secretory pathway in order to remodel its architecture and modulate the cellular secretion capacity. By contrast, membranes of the early secretory pathway, similar to the endosomal system, can also initiate and modulate signalling cascades, thereby spatially organising cellular signalling and eliciting a different cellular outcome than when signalling is localised to the plasma membrane. This Commentary highlights recent contributions to our understanding of the mutual regulation of the secretory pathway and cellular signalling.

Two Discontinuous Segments in the Carboxyl Terminus Are Required for Membrane Targeting of the Rat γ-Aminobutyric Acid Transporter-1 (GAT1)

Like all members of the Na+/Cl--dependent neurotransmitter transporter family, the rat γ-aminobutyric acid transporter-1 (GAT1) is sorted and targeted to specialized domains of the cell surface. Here we identify two discontinuous signals in the carboxyl terminus of GAT1 that cooperate to drive surface expression. This conclusion is based on the following observations. Upon deletion of the last 37 amino acids, the resulting GAT1-Δ37 remained trapped in the endoplasmic reticulum. The presence of 10 additional residues (GAT1-Δ27) sufficed to support the interaction with the coat protein complex II component Sec24D; surface expression of GAT1-Δ27 reached 50% of the wild type level. Additional extensions up to the position -3 (GAT1-Δ3) did not further enhance surface expression. Thus the last three amino acids (AYI) comprise a second distal signal. The sequence AYI is reminiscent of a type II PDZ-binding motif; accordingly substituting Glu for Ile abrogated the effect of this motif. Neither the AYI motif nor the last 10 residues rescued the protein from intracellular retention when grafted onto GAT1-Δ37 and GAT1-Δ32; the AYI motif was dispensable for targeting of GAT1 to the growth cone of differentiating PC12 cells. We therefore conclude that the two segments act in a hierarchical manner such that the proximal motif (569VMI571) supports endoplasmic reticulum export of the protein and the distal AYI motif places GAT1 under the control of the exocyst.

The Endoplasmic Reticulum Exit of Glutamate Transporter Is Regulated by the Inducible Mammalian Yip6b/GTRAP3-18 Protein

GTRAP3-18 interacts with and reduces the activity of the neuronal specific Na+/K+ glutamate transporter, EAAC1 both in vitro and in vivo. GTRAP3-18 and the related isoform, JM4, are distant relatives of the Rab GTPase-interacting factor PRA1, and share a topology of four transmembrane domains and cytosolic termini. GTRAP3-18 and JM4 are resident endoplasmic reticulum (ER) proteins. The physiological role of GTRAP3-18 is poorly understood. We demonstrate for the first time that GTRAP3-18 is a regulator of ER protein trafficking. Expression of GTRAP3-18 delays the ER exit of EAAC1, as well as other members of the excitatory amino acid transporter family. GTRAP3-18 uses hydrophobic domain interactions in the ER membrane to self-associate and cytoplasmic interactions at the C terminus to regulate trafficking. The features of GTRAP3-18 activity are consistent with recent phylogenic sequence analyses suggesting GTRAP3-18 and JM4 be reclassified as mammalian isoforms of the yeast protein family Yip, Yip6b, and Yip6a, respectively.

Genistein inhibits Vitamin D hydroxylases CYP24 and CYP27B1 expression in prostate cells

In human prostate cancer cells, the availability of the steroid hormone 1,25-dihydroxyvitamin D(3) for antimitotic action is determined through the activity of the two enzymes CYP24 and CYP27B1, viz. 25-hydroxyvitamin D-24-hydroxylase and 25-hydroxyvitamin D-1alpha-hydroxylase. High performance liquid chromatography (HPLC) analysis of [(3)H]25(OH)D(3) metabolism in human prostate cancer DU-145 cells revealed that genistein and other isoflavonoids, such as dihydrogenistein and daidzein, as well as the antiestrogenic compound ICI 182,780, inhibited Vitamin D-metabolizing enzyme activities. Reverse transcriptase-polymerase chain reaction (RT-PCR) showed that only in case of genistein this was due to transcriptional inhibition of CYP24 and CYP27B1 gene expressions. In case of CYP27B1, reduction of gene activity involves histone deacetylation because genistein was inactive in the presence of the histone deactylase inhibitor trichostatin A. In contrast, under the same condition, CYP24 gene activity was largely suppressed. In summary, our results suggest that a combined effect of genistein and trichostatin A could increase the responsiveness of human prostate cancer cells to the antiproliferative action of 1,25-dihydroxyvitamin D(3).

ERK7 is a negative regulator of protein secretion in response to amino‐acid starvation by modulating Sec16 membrane association

RNAi screening for kinases regulating the functional organization of the early secretory pathway in Drosophila S2 cells has identified the atypical Mitotic‐Associated Protein Kinase (MAPK) Extracellularly regulated kinase 7 (ERK7) as a new modulator. We found that ERK7 negatively regulates secretion in response to serum and amino‐acid starvation, in both Drosophila and human cells. Under these conditions, ERK7 turnover through the proteasome is inhibited, and the resulting higher levels of this kinase lead to a modification in a site within the C‐terminus of Sec16, a key ER exit site component. This post‐translational modification elicits the cytoplasmic dispersion of Sec16 and the consequent disassembly of the ER exit sites, which in turn results in protein secretion inhibition. We found that ER exit site disassembly upon starvation is TOR complex 1 (TORC1) independent, showing that under nutrient stress conditions, cell growth is not only inhibited at the transcriptional and translational levels, but also independently at the level of secretion by inhibiting the membrane flow through the early secretory pathway. These results reveal the existence of new signalling circuits participating in the complex regulation of cell growth.