Interaction Of RACK1 Research Articles

Regulation of CaV3.2 channels by the receptor for activated C kinase 1 (Rack-1).

This study describes the interaction between CaV3.2 calcium channels and the receptor for activated C kinase 1 (Rack-1), a scaffold protein which has recently been implicated in neuropathic pain. The coexpression of CaV3.2 and Rack-1 in tsA-201 cells led to a reduction in the magnitude of whole-cell CaV3.2 currents and CaV3.2 channel expression at the plasma membrane. Co-immunoprecipitations from transfected cells show the formation of a molecular protein complex between Cav3.2 channels and Rack-1. We determined that the interaction of Rack-1 occurs at the intracellular II-III loop and the C-terminus of the channel. Finally, the coexpression of PKCβII abolished the effect of Rack-1 on current densities. Altogether, our findings show that Rack-1 regulates CaV3.2-mediated calcium entry in a PKC-dependent manner.

RACK1 plays a critical role in mast cell secretion and Ca2+ mobilization by modulating F-actin dynamics.

Although RACK1 is known to act as a signaling hub in immune cells, its presence and role in mast cells (MCs) is undetermined. MC activation via antigen stimulation results in mediator release and is preceded by cytoskeleton reorganization and Ca2+ mobilization. In this study, we found that RACK1 was distributed throughout the MC cytoplasm both in vivo and in vitro. After RACK1 knockdown (KD), MCs were rounded, and the cortical F-actin was fragmented. Following antigen stimulation, in RACK1 KD MCs, there was a reduction in cortical F-actin, an increase in monomeric G-actin and a failure to organize F-actin. RACK1 KD also increased and accelerated degranulation. CD63+ secretory granules were localized in F-actin-free cortical regions in non-stimulated RACK1 KD MCs. Additionally, RACK1 KD increased antigen-stimulated Ca2+ mobilization, but attenuated antigen-stimulated depletion of ER Ca2+ stores and thapsigargin-induced Ca2+ entry. Following MC activation there was also an increase in interaction of RACK1 with Orai1 Ca2+-channels, β-actin and the actin-binding proteins vinculin and MyoVa. These results show that RACK1 is a critical regulator of actin dynamics, affecting mediator secretion and Ca2+ signaling in MCs. This article has an associated First Person interview with the first author of the paper.

Trafficking, localization and degradation of the Na+,HCO3\u2212 co-transporter NBCn1 in kidney and breast epithelial cells

The Na+;HCO3− co-transporter NBCn1 (SLC4A7) is a major regulator of intracellular pH yet its trafficking and turnover are essentially unstudied. Here, we used MDCK-II and MCF-7 cells to investigate these processes in epithelial cells. GFP-NBCn1 membrane localization was abolished by truncation of the full NBCn1 C-terminal tail (C-tail) yet did not require the C-terminal PDZ-binding motif (ETSL). Glutathione-S-Transferase-pulldown of the C-tail followed by mass spectrometry analysis revealed putative interactions with multiple sorting-, degradation- and retention factors, including the scaffolding protein RACK1. Pulldown of FLAG-tagged deletion constructs mapped the RACK1 interaction to the proximal NBCn1 C-tail. Proximity Ligation Assay and co-immunoprecipitation confirmed that native NBCn1 interacts with RACK1 in a cellular context. Consistent with a functional role of this complex, RACK1 knockdown reduced NBCn1 membrane localization without affecting total NBCn1 expression. Notably, only non-confluent cells exhibited detectable NBCn1-RACK1 plasma membrane co-localization, suggesting that RACK1 regulates the trafficking of NBCn1 to the membrane. Whereas total NBCn1 degradation was slow, with a half-life of more than 24 h, one-third of surface NBCn1 was constitutively endocytosed from the basolateral membrane within 60 min. This suggests that a fraction of NBCn1 exhibits recycling between the basolateral membrane and intracellular compartment(s). Our findings have important implications for understanding NBCn1 regulation as well as its dysregulation in disease.

The RNA-binding protein SERBP1 interacts selectively with the signaling protein RACK1

The RACK1 protein interacts with numerous proteins involved in signal transduction, the cytoskeleton, and mRNA splicing and translation. We used the 2-hybrid system to identify additional proteins interacting with RACK1 and isolated the RNA-binding protein SERBP1. SERPB1 shares amino acid sequence homology with HABP4 (also known as Ki-1/57), a component of the RNA spicing machinery that has been shown previously to interact with RACK1. Several different isoforms of SERBP1, generated by alternative mRNA splicing, interacted with RACK1 with indistinguishable interaction strength, as determined by a 2-hybrid beta-galactosidase assay. Analysis of deletion constructs of SERBP1 showed that the C-terminal third of the SERBP1 protein, which contains one of its two substrate sites for protein arginine N-methyltransferase 1 (PRMT1), is necessary and sufficient for it to interact with RACK1. Analysis of single amino acid substitutions in RACK1, identified in a reverse 2-hybrid screen, showed very substantial overlap with those implicated in the interaction of RACK1 with the cAMP-selective phosphodiesterase PDE4D5. These data are consistent with SERBP1 interacting selectively with RACK1, mediated by an extensive interaction surface on both proteins.

Over-expression of insulin-response element binding protein-1 (IRE-BP1) in mouse pancreatic islets increases expression of RACK1 and TCTP: Beta cell markers of high glucose sensitivity

BackgroundA targeted analysis of the 50kDa C-terminal fragment of insulin-response element binding protein-1 (IRE-BP1) activation of target genes through the insulin receptor substrate receptor/PI-3 kinase/Akt pathway has been demonstrated for the insulin growth factor-1 receptor. The broader effects of 50kDa C-terminal IRE-BP1 fragment over-expression on protein abundance in pancreatic islet beta cells have not been determined. ResultsLiquid-chromatography coupled to tandem mass spectrometry (LC-MS/MS) analyses of replicate lysates of pancreatic islets isolated from background strain animals and transgenic animals, overexpressing IRE-BP1 in pancreatic islet beta cells, demonstrated statistically significant increases in the expression of proteins involved in protein synthesis, endoplasmic reticulum (ER) stress and scaffolding proteins important for protein kinase C signaling; some of which were confirmed by immunoblot analyses. Bioinformatic analysis of protein expression network patterns suggested IRE-BP1 over-expression leads to protein expression patterns indicative of activation of functional protein networks utilized for protein post-translational modification, protein folding, and protein synthesis. Co-immunoprecipitation experiments demonstrate a novel interaction between two differentially regulated proteins receptor for activated protein kinase C (RACK1) and translationally controlled tumor protein (TCTP). ConclusionsProteomic analysis of IRE-BP1 over-expression in pancreatic islet beta cells suggest IRE-BP1 (a) directly or indirectly through establishing hyperglycemia results in increased expression of ribosomal proteins and markers of ER stress and (b) leads to the enhanced and previously un-described interaction of RACK1 and TCTP. SignificanceThis study identified C-terminal 50kDa domain of IRE-BP1 over-expression results in increased markers of ER-stress and a novel interaction between the scaffolding proteins RACK1 and TCTP.

RACK1 stabilises the activity of PP2A to regulate the transformed phenotype in mammary epithelial cells

Conflicting reports implicate the scaffolding protein RACK1 in the progression of breast cancer. RACK1 has been identified as a key regulator downstream of growth factor and adhesion signalling and as a direct binding partner of PP2A. Our objective was to further characterise the interaction between PP2A and RACK1 and to advance our understanding of this complex in breast cancer cells. We examined how the PP2A holoenzyme is assembled on the RACK1 scaffold in MCF-7 cells. We used immobilized peptide arrays representing the entire PP2A-catalytic subunit to identify candidate amino acids on the C subunit of PP2A that might be involved in binding of RACK1. We identified the RACK1 interaction sites on PP2A. Stable cell lines expressing PP2A with FR69/70AA, R214A and Y218F substitutions were generated and it was confirmed that the RACK1/PP2A interaction is essential to stabilise PP2A activity. We used Real-Time Cell Analysis and a series of assays to demonstrate that disruption of the RACK1/PP2A complex also reduces the adhesion, proliferation, migration and invasion of breast cancer cells and plays a role in maintenance of the cancer phenotype. This work has significantly advanced our understanding of the RACK1/PP2A complex and suggests a pro-carcinogenic role for the RACK1/PP2A interaction. This work suggests that approaches to target the RACK1/PP2A complex are a viable option to regulate PP2A activity and identifies a novel potential therapeutic target in the treatment of breast cancer.

Increased PKCα activity by Rack1 overexpression is responsible for chemotherapy resistance in T-cell acute lymphoblastic leukemia-derived cell line.

Chemoresistant mechanisms in T-cell acute lymphoblastic leukemia (T-ALL) patients are not clarified. The apoptotic signaling mediated by receptor of activated C kinase 1 (Rack1), protein kinase C (PKC) and FEM1 homolog b (FEM1b) was investigated in two T-ALL-derived cell lines (Jurkat and CCRF-CEM) following treatment with chemotherapy drugs vincristine and prednisone. Serum starvation or chemotherapeutic drugs significantly reduced Rack1 level and PKC activation, while promoted cellular apoptosis in both cell lines. Rack1 overexpression protected T-ALL cell against starvation or chemotherapeutic drug-induced apoptosis. Moreover, Rack1 overexpression reduced the level of cytochrome c and active caspase 3 as well as FEM1b and apoptotic protease activating factor-1 (Apaf-1), and inhibited induction of cellular apoptosis in chemotherapeutic drug-treated Jurkat cell. Interaction of Rack1 and PKCα, not PKCβ, was detected in both cell lines. Of note, Rack1 overexpression abrogated reduction of PKC kinase activity in chemotherapeutic drug-treated T-ALL cell. PKC kinase inhibitor Go6976 or siPKCα inhibited downregulation of FEM1b and/or Apaf-1, and thus increased cellular apoptosis in Rack1-overexpressed T-ALL cell receiving chemotherapeutic drugs. Accordingly, our data provided evidence that increased Rack1-mediated upregulation of PKC kinase activity may be responsible for the development of chemoresistance in T-ALL-derived cell line potentially by reducing FEM1b and Apaf-1 level.

Regulation of the ribosome and protein synthesis by RNAi

In many biological processes, e.g. the development of multicellular organisms, a tight regulation of the protein synthesis is a necessity. Among numerous mechanisms for translational control, RNA interference (RNAi) based mechanisms have been shown to regulate the translation of messenger RNA (mRNA) and hence regulate the synthesis of proteins. The key proteins in all RNAi mechanisms are the argonaute proteins. The only catalytically active argonaute protein is denoted argonaute2 (Ago2) in humans. This single chain protein is comprised of four globular domains arranged in a crescent shape tertiary structure [1]. The guide RNA binding specificity lies within the Mid and PAZ domain while the active site resides in the PIWI domain. In 2011 it was reported that the receptor for activated C-kinase (RACK1), an integral protein of the ribosomal 40S subunit, directly binds the microRNA induced silencing complex (miRISC) [2] and thereby contributes to gene repression through RNAi mediated knockdown. This interaction of RACK1 with miRISC was furthermore shown to be a specific interaction between Ago2 and RACK1. Structural investigation of this interaction will be of great interest to elucidate how Ago2 is positioned in relation to ribosome bound mRNA and if this positioning of Ago2 on the ribosome facilitates mRNA binding to the guide RNA bound in Ago2. In our studies we will co-crystallize recombinantly expressed Ago2 with 80S ribosome from S. cerevisiae [3] and solve the structure by x-ray crystallography. Recent project progress will be presented on the conference poster.

Extra-nuclear activity of INSM1 transcription factor enhances insulin receptor signaling pathway and Nkx6.1 expression through RACK1 interaction

INSM1 is an islet transcription factor essential for pancreas development. INSM1 functions as a transcriptional repressor of NeuroD/β2 and insulin gene in the pancreas. INSM1 also possesses extra-nuclear activities through binding to multiple cellular regulators such as cyclin D1 and RACK1. In this study, we report that the interaction of INSM1 and RACK1 is essential to enhance the insulin receptor (InR)-mediated signaling pathway. A proline-rich region in the N-terminus of INSM1 is required for RACK1 binding, which interrupts RACK1–InR interaction and enhances InR signal activation. Binding of INSM1 to RACK1 increases AKT phosphorylation. The INSM1-enhanced AKT phosphorylation can be inhibited by the PI3K inhibitor, LY294002. When INSM1 induces AR42J cell trans-differentiation, the Nkx6.1 gene is activated through the InR-mediated signaling pathway and an elevation of the acetyl-H4 modification on the Nkx6.1 gene promoter/enhancer is observed. The PI3K inhibitor interrupts Nkx6.1 and insulin gene expression. Therefore, we conclude that the extra-nuclear activity of INSM1 by enhancing the PI3K/AKT signaling pathway is important for pancreatic cell differentiation.

Trop‐2 inhibits prostate cancer cell adhesion to fibronectin through the β1 integrin‐RACK1 axis

Trop-2 is a transmembrane glycoprotein upregulated in several human carcinomas, including prostate cancer (PrCa). Trop-2 has been suggested to regulate cell-cell adhesion, given its high homology with the other member of the Trop family, Trop-1/EpCAM, and its ability to bind the tight junction proteins claudin-1 and claudin-7. However, a role for Trop-2 in cell adhesion to the extracellular matrix has never been postulated. Here, we show for the first time that Trop-2 expression in PrCa cells correlates with their aggressiveness. Using either shRNA-mediated silencing of Trop-2 in cells that endogenously express it, or ectopic expression of Trop-2 in cells that do not express it, we show that Trop-2 inhibits PrCa cell adhesion to fibronectin (FN). In contrast, expression of another transmembrane receptor, α(v) β(5) integrin, does not affect cell adhesion to this ligand. We find that Trop-2 does not modulate either protein or activation levels of the prominent FN receptors, β(1) integrins, but acts through increasing β(1) association with the adaptor molecule RACK1 and redistribution of RACK1 to the cell membrane. As a result of Trop-2 expression, we also observe activation of Src and FAK, known to occur upon β(1) -RACK1 interaction. These enhanced Src and FAK activities are not mediated by changes in either the activity of IGF-IR, which is known to bind RACK1, or IGF-IR's ability to associate with β(1) integrins. In summary, our data demonstrate that the transmembrane receptor Trop-2 is a regulator of PrCa cell adhesion to FN through activation of the β(1) integrin-RACK1-FAK-Src signaling axis.

The ribosomal protein RACK1 is required for microRNA function in both C. elegans and humans.

Despite the importance of microRNAs (miRNAs) in gene regulation, it is unclear how the miRNA-Argonaute complex--or miRNA-induced silencing complex (miRISC)--can regulate the translation of their targets in such diverse ways. We demonstrate here a direct interaction between the miRISC and the ribosome by showing that a constituent of the eukaryotic 40S subunit, receptor for activated C-kinase (RACK1), is important for miRNA-mediated gene regulation in animals. In vivo studies demonstrate that RACK1 interacts with components of the miRISC in nematodes and mammals. In both systems, the alteration of RACK1 expression alters miRNA function and impairs the association of the miRNA complex with the translating ribosomes. Our data indicate that RACK1 can contribute to the recruitment of miRISC to the site of translation, and support a post-initiation mode of miRNA-mediated gene repression.

RACK1 Associates with Muscarinic Receptors and Regulates M2 Receptor Trafficking

Receptor internalization from the cell surface occurs through several mechanisms. Some of these mechanisms, such as clathrin coated pits, are well understood. The M2 muscarinic acetylcholine receptor undergoes internalization via a poorly-defined clathrin-independent mechanism. We used isotope coded affinity tagging and mass spectrometry to identify the scaffolding protein, receptor for activated C kinase (RACK1) as a protein enriched in M2-immunoprecipitates from M2-expressing cells over those of non-M2 expressing cells. Treatment of cells with the agonist carbachol disrupted the interaction of RACK1 with M2. We further found that RACK1 overexpression inhibits the internalization and subsequent down regulation of the M2 receptor in a receptor subtype-specific manner. Decreased RACK1 expression increases the rate of agonist internalization of the M2 receptor, but decreases the extent of subsequent down-regulation. These results suggest that RACK1 may both interfere with agonist-induced sequestration and be required for subsequent targeting of internalized M2 receptors to the degradative pathway.

Phosphorylation of RACK1 on Tyrosine 52 by c-Abl Is Required for Insulin-like Growth Factor I-mediated Regulation of Focal Adhesion Kinase

Focal Adhesion Kinase (FAK) activity is controlled by growth factors and adhesion signals in tumor cells. The scaffolding protein RACK1 (receptor for activated C kinases) integrates insulin-like growth factor I (IGF-I) and integrin signaling, but whether RACK1 is required for FAK function is unknown. Here we show that association of FAK with RACK1 is required for both FAK phosphorylation and dephosphorylation in response to IGF-I. Suppression of RACK1 by small interfering RNA ablates FAK phosphorylation and reduces cell adhesion, cell spreading, and clonogenic growth. Peptide array and mutagenesis studies localize the FAK binding interface to blades I-III of the RACK1 beta-propeller and specifically identify a set of basic and hydrophobic amino acids (Arg-47, Tyr-52, Arg-57, Arg-60, Phe-65, Lys-127, and Lys-130) as key determinants for association with FAK. Mutation of tyrosine 52 alone is sufficient to disrupt interaction of RACK1 with FAK in cells where endogenous RACK1 is suppressed by small interfering RNA. Cells expressing a Y52F mutant RACK1 are impaired in adhesion, growth, and foci formation. Comparative analyses of homology models and crystal structures for RACK1 orthologues suggest a role for Tyr-52 as a site for phosphorylation that induces conformational change in RACK1, switching the protein into a FAK binding state. Tyrosine 52 is further shown to be phosphorylated by c-Abl kinase, and the c-Abl inhibitor STI571 disrupts FAK interaction with RACK1. We conclude that FAK association with RACK1 is regulated by phosphorylation of Tyr-52. Our data reveal a novel mechanism whereby IGF-I and c-Abl control RACK1 association with FAK to facilitate adhesion signaling.

Filamin‐A modulates CFTR surface expression via Filamin‐A Ig repeat 21 and interacts with the scaffold protein RACK1

Filamin‐A (FlnA) binds to CFTR through a protein‐protein interaction that is disrupted by the missense mutation S13F in CFTR, causing unstable surface expression of CFTR. We hypothesized that the N‐terminal sequence of CFTR binds to immunoglobulin repeat 21 of FlnA (Ig21), based on previously studied binding preferences of Ig21. We confirmed binding of a CFTR N‐terminal peptide to Ig21 and solved the structure of the peptide bound to Ig21 in a novel staggered 2:1 complex, in which the single CFTR N‐terminal peptide completes an extended beta sheet spanning two parallel Ig21 domains. The structure reveals how the disease‐causing CF mutation S13F disrupts the FlnA:CFTR interaction. To test functional effects of Ig21, we delivered Ig21 into Calu‐3 human airway epithelial cells and examined CFTR apical membrane expression by biotinylation. Ig21 caused a dose‐dependent decrease in biotinylated CFTR. CoIP experiments demonstrated an interaction between CFTR and FlnA. We also detected RACK1 in IPs of FlnA and vice versa and colocalization of RACK1 and FlnA by IF. To study RACK1 binding to FlnA, FlnA or CFTR were IPed from Calu‐3 cells, washed under high stringent conditions, and incubated with recombinant RACK1. RACK1 was shown to bind to FlnA in a dose dependent manner, but not to CFTR. Our results suggest that the important FlnA:CFTR interaction is indeed mediated by FlnA‐Ig21 and that FlnA:RACK1 interaction may be important for RACK1‐mediated recruitment of PKCε to CFTR. Supported by the Ralph Wilson Medical Research Foundation (SM), NIH R01‐58598 and ‐67190 (CML) and an RDP grant from the Cystic Fibrosis Foundation (CML).

1H NMR structural and functional characterisation of a cAMP-specific phosphodiesterase-4D5 (PDE4D5) N-terminal region peptide that disrupts PDE4D5 interaction with the signalling scaffold proteins, βarrestin and RACK1

The unique 88 amino acid N-terminal region of cAMP-specific phosphodiesterase-4D5 (PDE4D5) contains overlapping binding sites conferring interaction with the signaling scaffold proteins, βarrestin and RACK1. A 38-mer peptide, whose sequence reflected residues 12 through 49 of PDE4D5, encompasses the entire N-terminal RACK1 Interaction Domain (RAID1) together with a portion of the βarrestin binding site. 1H NMR and CD analyses indicate that this region has propensity to form a helical structure. The leucine-rich hydrophobic grouping essential for RACK1 interaction forms a discrete hydrophobic ridge located along a single face of an amphipathic α-helix with Arg34 and Asn36, which also play important roles in RACK1 binding. The Asn22/Pro23/Trp24/Asn26 grouping, essential for RACK1 interaction, was located at the N-terminal head of the amphipathic helix that contained the hydrophobic ridge. RAID1 is thus provided by a distinct amphipathic helical structure. We suggest that the binding of PDE4D5 to the WD-repeat protein, RACK1, may occur in a manner akin to the helix–helix interaction shown for Gγ binding to the WD-repeat protein, Gβ. A more extensive section of the PDE4D5 N-terminal sequence (Thr11-Ala85) is involved in βarrestin binding. Several residues within the RAID1 helix contribute to this interaction however. We show here that these residues form a focused band around the centre of the RAID1 helix, generating a hydrophobic patch (from Leu29, Val30 and Leu33) flanked by polar/charged residues (Asn26, Glu27, Asp28, Arg34). The interaction with βarrestin exploits a greater circumference on the RAID1 helix, and involves two residues (Glu27, Asp28) that do not contribute to RACK1 binding. In contrast, the interaction of RACK1 with RAID1 is extended over a greater length of the helix and includes Leu37/Leu38, which do not contribute to βarrestin binding. A membrane-permeable, stearoylated Val12-Ser49 38-mer peptide disrupted the interaction of both βarrestin and RACK1 with endogenous PDE4D5 in HEKB2 cells, whilst a cognate peptide with a Glu27Ala substitution selectively failed to disrupt PDE4D5/RACK1 interaction. The stearoylated Val12-Ser49 38-mer peptide enhanced the isoprenaline-stimulated PKA phosphorylation of the β2-adrenergic receptors (β2AR) and its activation of ERK, whilst the Glu27Ala peptide was ineffective in both these regards.

Receptor for Activated C-Kinase 1, a Novel Interaction Partner of Type II Bone Morphogenetic Protein Receptor, Regulates Smooth Muscle Cell Proliferation in Pulmonary Arterial Hypertension

Pulmonary arterial hypertension (PAH) is characterized by selective elevation of pulmonary arterial pressure. The pathological hallmark of PAH is the narrowing of pulmonary arterioles secondary to endothelial cell dysfunction and smooth muscle cell proliferation. Heterozygous mutations in BMPR2, encoding the type II bone morphogenetic protein receptor (BMPRII), were identified in PAH, suggesting that alterations to BMPRII function are involved in disease onset and/or progression. We identified the receptor for activated C-kinase (RACK1) as a novel interaction partner of BMPRII by yeast 2-hybrid analyses using the kinase domain of BMPRII as a bait. Glutathione-S-transferase pull-down and coimmunoprecipitation confirmed the interaction of RACK1 with BMPRII in vitro and in vivo. RACK1-BMPRII interaction was reduced when kinase domain mutations occurring in patients with PAH were introduced to BMPRII. Immunohistochemistry of lung sections from PAH and control patients and immunofluorescence analysis of primary pulmonary arterial smooth muscle cells demonstrated colocalization of BMPRII and RACK1 in vivo. Quantitative reverse-transcription polymerase chain reaction and Western blot analysis showed significant downregulation of RACK1 expression in the rat model of monocrotaline-induced PAH but not in pulmonary arterial smooth muscle cells from PAH patients. Abrogation of RACK1 expression in pulmonary arterial smooth muscle cells led to decreased Smad1 phosphorylation and increased proliferation, whereas overexpression of RACK1 led to increased Smad1 phosphorylation and decreased proliferation. RACK1, a novel interaction partner of BMPRII, constitutes a new negative regulator of pulmonary arterial smooth muscle cell proliferation, suggesting a potential role for RACK1 in the pathogenesis of PAH.

Peptide modulators of Src activity in G1 regulate entry into S phase and proliferation of NIH 3T3 cells

Cascades of kinases and phosphatases are regulated by selective protein–protein interactions that are essential for signal transduction. Peptide modulators of these interactions have been used to dissect the function of individual components of the signaling cascade, without relying on either the over- or underexpression of proteins. Previously, we identified RACK1 as an endogenous substrate, binding partner and inhibitor of Src tyrosine kinases. Here, we utilized cell-permeable peptides that selectively disrupt or enhance the interaction of RACK1 and Src to further examine the function of RACK1. Our results provide direct physiologic evidence that RACK1 regulates growth of NIH3T3 cells by suppressing the activity of Src and other cell cycle regulators in G1, and delaying entry into S phase. They also demonstrate the potential for using peptide modulators of Src activity as a tool for regulating cell growth, and for designing new strategies for cancer therapy that target specific protein–protein interactions.

RACK1 inhibits colonic cell growth by regulating Src activity at cell cycle checkpoints

Previously, we showed that Src tyrosine kinases are activated early in the development of human colon cancer and are suppressed as intestinal cells differentiate. We identified RACK1 as an endogenous substrate, binding partner and inhibitor of Src. Here we show (by overexpressing RACK1, depleting Src or RACK1 and utilizing cell-permeable peptides that perturb RACK1's interaction with Src) that RACK1 regulates growth of colon cells by suppressing Src activity at G(1) and mitotic checkpoints, and consequently delaying cell cycle progression. Activated Src rescues RACK1-inhibited growth of HT-29 cells. Conversely, inhibiting Src abolishes growth promoted by RACK1 depletion in normal cells. Two potential mechanisms whereby RACK1 regulates mitotic exit are identified: suppression of Src-mediated Sam68 phosphorylation and maintenance of the cyclin-dependent kinase (CDK) 1-cyclin B complex in an active state. Our results reveal novel mechanisms of cell cycle control in G(1) and mitosis of colon cells. The significance of this work lies in the discovery of a mechanism by which the growth of colon cancer cells can be slowed, by RACK1 suppression of an oncogenic kinase at critical cell cycle checkpoints. Small molecules that mimic RACK1 function may provide a powerful new approach to the treatment of colon cancer.

RACK1 regulates Src-mediated Sam68 and p190RhoGAP signaling.

RACK1 is the founding member of a family of receptors for activated C kinase collectively called RACKs. Upon activation of PKC, RACK1 co-localizes with the Src tyrosine kinase at the plasma membrane and functions as a substrate, binding partner and inhibitor of Src (as measured in vitro), and a growth inhibitor in NIH 3T3 cells. To further analyze the function of RACK1 in Src and PKC signaling, we utilized cell-permeable peptides that modulate the interaction of RACK1 and betaIIPKC, thereby affecting betaIIPKC translocation and function. We found that the association of betaIIPKC and RACK1 is necessary for Src phosphorylation of RACK1. Src activity is required for tyrosine phosphorylation of RACK1, and for RACK1 binding to Src, but not to betaIIPKC. Endogenous Src kinase activity, as measured by phosphorylation of Sam68 (a mitotic-specific Src substrate involved in cell cycle regulation and RNA splicing) or p190RhoGAP (a Src substrate and GTPase-activating protein involved in actin reorganization), increases with disruption of the Src-RACK1 complex, and decreases with enhanced complex formation. RACK1 inhibits Src-mediated p190RhoGAP signaling and actin cytoskeleton rearrangement. Thus, RACK1 functions as an endogenous inhibitor of the Src kinase in diverse signaling pathways that regulate distinct cellular functions. Our results demonstrate the potential for using peptide modulators of Src activity as a tool for uncovering the function of Src in cells.

Delineation of RAID1, the RACK1 interaction domain located within the unique N-terminal region of the cAMP-specific phosphodiesterase, PDE4D5

BackgroundThe cyclic AMP specific phosphodiesterase, PDE4D5 interacts with the β-propeller protein RACK1 to form a signaling scaffold complex in cells. Two-hybrid analysis of truncation and mutant constructs of the unique N-terminal region of the cAMP-specific phosphodiesterase, PDE4D5 were used to define a domain conferring interaction with the signaling scaffold protein, RACK1.ResultsTruncation and mutagenesis approaches showed that the RACK1-interacting domain on PDE4D5 comprised a cluster of residues provided by Asn-22/Pro-23/Trp-24/Asn-26 together with a series of hydrophobic amino acids, namely Leu-29, Val-30, Leu-33, Leu-37 and Leu-38 in a 'Leu-Xaa-Xaa-Xaa-Leu' repeat. This was done by 2-hybrid analyses and then confirmed in biochemical pull down analyses using GST-RACK1 and mutant PDE4D5 forms expressed in COS cells. Mutation of Arg-34, to alanine, in PDE4D5 attenuated its interaction with RACK1 both in 2-hybrid screens and in pull down analyses. A 38-mer peptide, whose sequence reflected residues 12 through 49 of PDE4D5, bound to RACK1 with similar affinity to native PDE4D5 itself (Ka circa 6 nM).ConclusionsThe RACK1 Interaction Domain on PDE4D5, that we here call RAID1, is proposed to form an amphipathic helical structure that we suggest may interact with the C-terminal β-propeller blades of RACK1 in a manner akin to the interaction of the helical G-γ signal transducing protein with the β-propeller protein, G-β.