Dissecting Gq/11-Mediated Plasma Membrane Translocation of Sphingosine Kinase-1.

Diverse extracellular signals induce plasma membrane translocation of sphingosine kinase-1 (SphK1), thereby enabling inside-out signaling of sphingosine-1-phosphate. We have shown before that Gq-coupled receptors and constitutively active Gαq/11 specifically induced a rapid and long-lasting SphK1 translocation, independently of canonical Gq/phospholipase C (PLC) signaling. Here, we further characterized Gq/11 regulation of SphK1. SphK1 translocation by the M3 receptor in HEK-293 cells was delayed by expression of catalytically inactive G-protein-coupled receptor kinase-2, p63Rho guanine nucleotide exchange factor (p63RhoGEF), and catalytically inactive PLCß3, but accelerated by wild-type PLCß3 and the PLCδ PH domain. Both wild-type SphK1 and catalytically inactive SphK1-G82D reduced M3 receptor-stimulated inositol phosphate production, suggesting competition at Gαq. Embryonic fibroblasts from Gαq/11 double-deficient mice were used to show that amino acids W263 and T257 of Gαq, which interact directly with PLCß3 and p63RhoGEF, were important for bradykinin B2 receptor-induced SphK1 translocation. Finally, an AIXXPL motif was identified in vertebrate SphK1 (positions 100-105 in human SphK1a), which resembles the Gαq binding motif, ALXXPI, in PLCß and p63RhoGEF. After M3 receptor stimulation, SphK1-A100E-I101E and SphK1-P104A-L105A translocated in only 25% and 56% of cells, respectively, and translocation efficiency was significantly reduced. The data suggest that both the AIXXPL motif and currently unknown consequences of PLCß/PLCδ(PH) expression are important for regulation of SphK1 by Gq/11.

CIB1: a small protein with big ambitions.

Calcium- and integrin-binding protein 1 (CIB1) is a small, ubiquitously expressed protein that was first identified as an intracellular binding partner of a platelet-specific α-integrin cytoplasmic tail. Although early studies revealed a role for CIB1 in regulating platelet integrin activity, recent studies have indicated a more diverse role for CIB1 in many different cell types and processes, including calcium signaling, migration, adhesion, proliferation, and survival. Increasing evidence also points to a novel role for CIB1 in cancer and cardiovascular disease. In addition, an array of CIB1 binding partners has been identified that provide important insight into how CIB1 may regulate these processes. Some of these binding partners include the serine/threonine kinases, p21-activated kinase 1 (PAK1), apoptosis signal-regulating kinase 1 (ASK1), and polo-like kinase 3 (PLK3). Structural and mutational studies indicate that CIB1 binds most or all of its partners via a well-defined hydrophobic cleft. Although CIB1 itself lacks known enzymatic activity, it supports the PI3K/AKT and MEK/ERK oncogenic signaling pathways, in part, by directly modulating enzymes in these pathways. In this review, we discuss our current understanding of CIB1 and key questions regarding structure and function and how this seemingly diminutive protein impacts important signaling pathways and cellular processes in human health and disease.-Leisner, T. M., Freeman, T. C., Black, J. L., Parise, L. V. CIB1: a small protein with big ambitions.

CIB1 depletion impairs cell survival and tumor growth in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with generally poor prognosis and no available targeted therapies, highlighting a critical unmet need to identify and characterize novel therapeutic targets. We previously demonstrated that CIB1 is necessary for cancer cell survival and proliferation via regulation of two oncogenic signaling pathways, RAF-MEK-ERK and PI3K-AKT. Because these pathways are often upregulated in TNBC, we hypothesized that CIB1 may play a broader role in TNBC cell survival and tumor growth. Methods utilized include inducible RNAi depletion of CIB1 in vitro and in vivo, immunoblotting, clonogenic assay, flow cytometry, RNA-sequencing, bioinformatics analysis, and Kaplan-Meier survival analysis. CIB1 depletion resulted in significant cell death in 8 of 11 TNBC cell lines tested. Analysis of components related to PI3K-AKT and RAF-MEK-ERK signaling revealed that elevated AKT activation status and low PTEN expression were key predictors of sensitivity to CIB1 depletion. Furthermore, CIB1 knockdown caused dramatic shrinkage of MDA-MB-468 xenograft tumors in vivo. RNA sequence analysis also showed that CIB1 depletion in TNBC cells activates gene programs associated with decreased proliferation and increased cell death. CIB1 expression levels per se did not predict TNBC susceptibility to CIB1 depletion, and CIB1 mRNA expression levels did not associate with TNBC patient survival. Our data are consistent with the emerging concept of non-oncogene addiction, where a large subset of TNBCs depend on CIB1 for cell survival and tumor growth, independent of CIB1 expression levels. Our data establish CIB1 as a novel therapeutic target for TNBC.

Pronunciation difficulty, temporal regularity, and the speech-to-song illusion.

The speech-to-song illusion (Deutsch et al., 2011) tracks the perceptual transformation from speech to song across repetitions of a brief spoken utterance. Because it involves no change in the stimulus itself, but a dramatic change in its perceived affiliation to speech or to music, it presents a unique opportunity to comparatively investigate the processing of language and music. In this study, native English-speaking participants were presented with brief spoken utterances that were subsequently repeated ten times. The utterances were drawn either from languages that are relatively difficult for a native English speaker to pronounce, or languages that are relatively easy for a native English speaker to pronounce. Moreover, the repetition could occur at regular or irregular temporal intervals. Participants rated the utterances before and after the repetitions on a 5-point Likert-like scale ranging from "sounds exactly like speech" to "sounds exactly like singing." The difference in ratings before and after was taken as a measure of the strength of the speech-to-song illusion in each case. The speech-to-song illusion occurred regardless of whether the repetitions were spaced at regular temporal intervals or not; however, it occurred more readily if the utterance was spoken in a language difficult for a native English speaker to pronounce. Speech circuitry seemed more liable to capture native and easy-to-pronounce languages, and more reluctant to relinquish them to perceived song across repetitions.

Identification of novel integrin binding partners for calcium and integrin binding protein 1 (CIB1): structural and thermodynamic basis of CIB1 promiscuity.

The short cytoplasmic tails of the α- and ß-chains of integrin adhesion receptors regulate integrin activation and cell signaling. Significantly less is known about proteins that bind to α-integrin cytoplasmic tails (CTs) as opposed to ß-CTs to regulate integrins. Calcium and integrin binding protein 1 (CIB1) was previously identified as an αIIb binding partner that inhibits agonist-induced activation of the platelet-specific integrin, αIIbß3. A sequence alignment of all α-integrin CTs revealed that key residues in the CIB1 binding site of αIIb are well-conserved, and was used to delineate a consensus binding site (I/L-x-x-x-L/M-W/Y-K-x-G-F-F). Because the CIB1 binding site of αIIb is conserved in all α-integrins and CIB1 expression is ubiquitous, we asked if CIB1 could interact with other α-integrin CTs. We predicted that multiple α-integrin CTs were capable of binding to the same hydrophobic binding pocket on CIB1 with docking models generated by all-atom replica exchange discrete molecular dynamics. After demonstrating novel in vivo interactions between CIB1 and other whole integrin complexes with co-immunoprecipitations, we validated the modeled predictions with solid-phase competitive binding assays, which showed that other α-integrin CTs compete with the αIIb CT for binding to CIB1 in vitro. Isothermal titration calorimetry measurements indicated that this binding is driven by hydrophobic interactions and depends on residues in the CIB1 consensus binding site. These new mechanistic details of CIB1-integrin binding imply that CIB1 could bind to all integrin complexes and act as a broad regulator of integrin function.

Chemoproteomic discovery of AADACL1 as a regulator of human platelet activation.

A comprehensive knowledge of the platelet proteome is necessary for understanding thrombosis and for envisioning antiplatelet therapies. To discover other biochemical pathways in human platelets, we screened platelets with a carbamate library designed to interrogate the serine hydrolase subproteome and used competitive activity-based protein profiling to map the targets of active carbamates. We identified an inhibitor that targets arylacetamide deacetylase-like 1 (AADACL1), a lipid deacetylase originally identified in invasive cancers. Using this compound, along with highly selective second-generation inhibitors of AADACL1, metabolomics, and RNA interference, we show that AADACL1 regulates platelet aggregation, thrombus growth, RAP1 and PKC activation, lipid metabolism, and fibrinogen binding to platelets and megakaryocytes. These data provide evidence that AADACL1 regulates platelet and megakaryocyte activation and highlight the value of this chemoproteomic strategy for target discovery in platelets.

Transgene delivery via intracellular electroporetic nanoinjection.

Development of an effective cytoplasmic delivery technique has remained an elusive goal for decades despite the success of pronuclear microinjection. Cytoplasmic injections are faster and easier than pronuclear injection and do not require the pronuclei to be visible; yet previous attempts to develop cytoplasmic injection have met with limited success. In this work we report a cytoplasmic delivery method termed intracellular electroporetic nanoinjection (IEN). IEN is unique in that it manipulates transgenes using electrical forces. The microelectromechanical system (MEMS) uses electrostatic charge to physically pick up transgenes and place them in the cytoplasm. The transgenes are then propelled through the cytoplasm and electroporated into the pronuclei using electrical pulses. Standard electroporation of whole embryos has not resulted in transgenic animals, but the MEMS device allows localized electroporation to occur within the cytoplasm for transgene delivery from the cytoplasm to the pronucleus. In this report we describe the principles which allow localized electroporation of the pronuclei including: the location of mouse pronuclei between 21 and 28 h post-hCG treatment, modeling data predicting the voltages needed for localized electroporation of pronuclei, and data on electric-field-driven movement of transgenes. We further report results of an IEN versus microinjection comparative study in which IEN produced transgenic pups with viability, transgene integration, and expression rates statistically comparable to microinjection. The ability to perform injections without visualizing or puncturing the pronuclei will widely benefit transgenic research, and will be particularly advantageous for the production of transgenic animals with embryos exhibiting reduced pronuclear visibility.