The very C-terminus of PRK1/PKN is essential for its activation by RhoA and downstream signaling

PRK1 is a lipid- and Rho GTPase-activated serine/threonine protein kinase implicated in the regulation of receptor trafficking, cytoskeletal dynamics and tumorigenesis. Although Rho binding has been mapped to the HR1 region in the regulatory domain of PRK1, the mechanism involved in the control of PRK1 activation following Rho binding is poorly understood. We now provide the first evidence that the very C-terminus beyond the hydrophobic motif in PRK1 is essential for the activation of this kinase by RhoA. Deletion of the HR1 region did not completely abolish the binding of PRK1-ΔHR1 to GTPγS-RhoA nor the activation of this mutant by GTPγS-RhoA in vitro. In contrast, removing of the last six amino acid residues from the C-terminus of PRK1 or truncating of a single C-terminal residue from PRK1-ΔHR1 completely abrogated the activation of these mutants by RhoA both in vitro and in vivo. The critical dependence of the very C-terminus of PRK1 on the signaling downstream of RhoA was further demonstrated by the failure of the PRK1 mutant lacking its six C-terminal residues to augment lisophosphatidic acid-elicited neurite retraction in neuronal cells. Thus, we show that the HR1 region is necessary but not sufficient in eliciting a full activation of PRK1 upon binding of RhoA. Instead, such activation is controlled by the very C-terminus of PRK1. Our results also suggest that the very C-terminus of PRK1, which is the least conserved among members of the protein kinase C superfamily, is a potential drug target for pharmacological intervention of RhoA-mediated signaling pathways

Src homology 3-domain growth factor receptor-bound 2-like (endophilin) interacting protein 1, a novel neuronal protein that regulates energy balance

To identify genes involved in the central regulation of energy balance, we compared hypothalamic mRNA from lean and obese Psammomys obesus, a polygenic model of obesity, using differential display PCR. One mRNA transcript was observed to be elevated in obese, and obese diabetic, P. obesus compared with lean animals and was subsequently found to be increased 4-fold in the hypothalamus of lethal yellow agouti (Ay/a) mice, a murine model of obesity and diabetes. Intracerebroventricular infusion of antisense oligonucleotide targeted to this transcript selectively suppressed its hypothalamic mRNA levels and resulted in loss of body weight in both P. obesus and Sprague Dawley rats. Reductions in body weight were mediated by profoundly reduced food intake without a concomitant reduction in metabolic rate. Yeast two-hybrid screening, and confirmation in mammalian cells by bioluminescence resonance energy transfer analysis, demonstrated that the protein it encodes interacts with endophilins, mediators of synaptic vesicle recycling and receptor endocytosis in the brain. We therefore named this transcript Src homology 3-domain growth factor receptor-bound 2-like (endophilin) interacting protein 1 (SGIP1). SGIP1 encodes a large proline-rich protein that is expressed predominantly in the brain and is highly conserved between species. Together these data suggest that SGIP1 is an important and novel member of the group of neuronal molecules required for the regulation of energy homeostasis.

Heterotrimeric G protein γ subunits provide functional selectivity in Gβγ dimer signaling in arabidopsis

The Arabidopsis thaliana heterotrimeric G protein complex is encoded by single canonical Galpha and Gbeta subunit genes and two Ggamma subunit genes (AGG1 and AGG2), raising the possibility that the two potential G protein complexes mediate different cellular processes. Mutants with reduced expression of one or both Ggamma genes revealed specialized roles for each Ggamma subunit. AGG1-deficient mutants, but not AGG2-deficient mutants, showed impaired resistance against necrotrophic pathogens, reduced induction of the plant defensin gene PDF1.2, and decreased sensitivity to methyl jasmonate. By contrast, both AGG1- and AGG2-deficient mutants were hypersensitive to auxin-mediated induction of lateral roots, suggesting that Gbetagamma1 and Gbetagamma2 synergistically inhibit auxin-dependent lateral root initiation. However, the involvement of each Ggamma subunit in this root response differs, with Gbetagamma1 acting within the central cylinder, attenuating acropetally transported auxin signaling, while Gbetagamma2 affects the action of basipetal auxin and graviresponsiveness within the epidermis and/or cortex. This selectivity also operates in the hypocotyl. Selectivity in Gbetagamma signaling was also found in other known AGB1-mediated pathways. agg1 mutants were hypersensitive to glucose and the osmotic agent mannitol during seed germination, while agg2 mutants were only affected by glucose. We show that both Ggamma subunits form functional Gbetagamma dimers and that each provides functional selectivity to the plant heterotrimeric G proteins, revealing a mechanism underlying the complexity of G protein-mediated signaling in plants.

A critical role for the short intracellular C terminus in receptor activity-modifying protein function

Receptor activity-modifying proteins (RAMPs) interact with and modify the behavior of the calcitonin receptor (CTR) and calcitonin receptor-like receptor (CLR). We have examined the contribution of the short intracellular C terminus, using constructs that delete the last eight amino acids of each RAMP. C-Terminal deletion of individual RAMPs had little effect on the signaling profile induced when complexed with CLR in COS-7 or human embryonic kidney (HEK)293 cells. Likewise, confocal microscopy revealed each of the mutant RAMPs translocated hemagglutinin-tagged CLR to the cell surface. In contrast, a pronounced effect of RAMP C-terminal truncation was seen for RAMP/CTRa complexes, studied in COS-7 cells, with significant attenuation of amylin receptor phenotype induction that was stronger for RAMP1 and -2 than RAMP3. The loss of amylin binding upon C-terminal deletion could be partially recovered with overexpression of Gαs, suggesting an impact of the RAMP C terminus on coupling of G proteins to the receptor complex. In HEK293 cells the c-Myc-RAMP1 C-terminal deletion mutant showed high receptor-independent cell surface expression; however, this construct showed low cell surface expression when expressed alone in COS-7 cells, indicating interaction of RAMPs with other cellular components via the C terminus. This mutant also had reduced cell surface expression when coexpressed with CTR. Thus, this study reveals important functionality of the RAMP C-terminal domain and identifies key differences in the role of the RAMP C terminus for CTR versus CLR-based receptors.

The regulation and function of the striated muscle activator of rho signaling (STARS) protein

Healthy living throughout the lifespan requires continual growth and repair of cardiac, smooth, and skeletal muscle. To effectively maintain these processes muscle cells detect extracellular stress signals and efficiently transmit them to activate appropriate intracellular transcriptional programs. The striated muscle activator of Rho signaling (STARS) protein, also known as Myocyte Stress-1 (MS1) protein and Actin-binding Rho-activating protein (ABRA) is highly enriched in cardiac, skeletal, and smooth muscle. STARS binds actin, co-localizes to the sarcomere and is able to stabilize the actin cytoskeleton. By regulating actin polymerization, STARS also controls an intracellular signaling cascade that stimulates the serum response factor (SRF) transcriptional pathway; a pathway controlling genes involved in muscle cell proliferation, differentiation, and growth. Understanding the activation, transcriptional control and biological roles of STARS in cardiac, smooth, and skeletal muscle, will improve our understanding of physiological and pathophysiological muscle development and function.

Genetic variation in SH3-domain GRB2-like (endophilin)-interacting protein 1 has a major impact on fat mass

Objective:

The SH3-domain GRB2-like (endophilin)-interacting protein 1 (SGIP1) gene has been shown to be differentially expressed in the hypothalamus of lean versus obese Israeli sand rats (Psammomys obesus), and is suspected of having a role in regulating food intake. The purpose of this study was to assess the role of genetic variation in SGIP1 in human disease.
Subjects:

We performed single-nucleotide polymorphism (SNP) genotyping in a large family pedigree cohort from the island of Mauritius. The Mauritius Family Study (MFS) consists of 400 individuals from 24 Indo-Mauritian families recruited from the genetically homogeneous population of Mauritius. We measured markers of the metabolic syndrome, including diabetes and obesity-related phenotypes such as fasting plasma glucose, waist:hip ratio, body mass index and fat mass.
Results:

Statistical genetic analysis revealed associations between SGIP1 polymorphisms and fat mass (in kilograms) as measured by bioimpedance. SNP genotyping identified associations between several genetic variants and fat mass, with the strongest association for rs2146905 (P=4.7 × 10−5). A strong allelic effect was noted for several SNPs where fat mass was reduced by up to 9.4% for individuals homozygous for the minor allele.
Conclusions:

Our results show association between genetic variants in SGIP1 and fat mass. We provide evidence that variation in SGIP1 is a potentially important determinant of obesity-related traits in humans.

A truncated fragment of Src protein kinase generated by calpain-mediated cleavage is a mediator of neuronal death in excitotoxicity

Excitotoxicity resulting from overstimulation of glutamate receptors is a major cause of neuronal death in cerebral ischemic stroke. The overstimulated ionotropic glutamate receptors exert their neurotoxic effects in part by overactivation of calpains, which induce neuronal death by catalyzing limited proteolysis of specific cellular proteins. Here, we report that in cultured cortical neurons and in vivo in a rat model of focal ischemic stroke, the tyrosine kinase Src is cleaved by calpains at a site in the N-terminal unique domain. This generates a truncated Src fragment of ?52 kDa, which we localized predominantly to the cytosol. A cell membrane-permeable fusion peptide derived from the unique domain of Src prevents calpain from cleaving Src in neurons and protects against excitotoxic neuronal death. To explore the role of the truncated Src fragment in neuronal death, we expressed a recombinant truncated Src fragment in cultured neurons and examined how it affects neuronal survival. Expression of this fragment, which lacks the myristoylation motif and unique domain, was sufficient to induce neuronal death. Furthermore, inactivation of the prosurvival kinase Akt is a key step in its neurotoxic signaling pathway. Because Src maintains neuronal survival, our results implicate calpain cleavage as a molecular switch converting Src from a promoter of cell survival to a mediator of neuronal death in excitotoxicity. Besides unveiling a new pathological action of Src, our discovery of the neurotoxic action of the truncated Src fragment suggests new therapeutic strategies with the potential to minimize brain damage in ischemic stroke.