Guanylate Kinase Domain Research Articles

Calorimetric and NMR Characterization of a Rem2 G-Domain Interaction with the Sh3-GK Core of the Ca2+ Channel Beta4 Subunit

The RGK (Rad, Rem1, Rem2 and Gem/Kir) subfamily of small GTPases has potent inhibitory effects on high voltage-activated Ca2+ channels. The inhibition has been shown to occur through a mechanism that is dependent on the Ca2+ channel beta subunit. In this study, we characterized an interaction between the G domain of Rem2 and the beta4 guanylate kinase (GK) domain using NMR and isothermal titration calorimetry (ITC) techniques. We determined that the interaction is endothermic, has a stoichiometry of 1, and a Kd 156 uM, which is three orders of magnitude weaker than the beta4 interaction with the pore-forming alpha1 subunit. NMR chemical shift perturbation analysis reveals that Rem2 interacts exclusively with the GK domain of the SH3-GK core by way of residues located in the former GMP binding region of the inactive kinase. The site is composed of residues just proximal to 310 helix sequences 2 and 3, and residues in alpha helix 6. Thus, the RGK binding site is clearly distinct from the binding site for the alpha1 subunit AID peptide. Site-directed mutagenesis of surface residues combined with ITC indicate that multiple GK sequences contribute to Rem2 binding collectively. These results support the idea that Ca2+ channel inhibition by Rem2 is unlikely to be the result of direct competition with alpha1 binding to the beta subunit. Instead, it argues in favor of a mechanism in which beta subunit-anchored Rem2 executes its inhibitory effect through other contacts with alpha1 subunits. This study demonstrates that NMR combined with ITC can provide detailed information on important weak protein-protein interactions involved in Ca2+ channel regulation.

Synaptic State-Dependent Functional Interplay between Postsynaptic Density-95 and Synapse-Associated Protein 102

Activity-dependent regulation of AMPA receptor (AMPAR)-mediated synaptic transmission is the basis for establishing differences in synaptic weights among individual synapses during developmental and experience-dependent synaptic plasticity. Synaptic signaling scaffolds of the Discs large (DLG)-membrane-associated guanylate kinase (MAGUK) protein family regulate these processes by tethering signaling proteins to receptor complexes. Using a molecular replacement strategy with RNAi-mediated knockdown in rat and mouse hippocampal organotypic slice cultures, a postsynaptic density-95 (PSD-95) knock-out mouse line and electrophysiological analysis, our current study identified a functional interplay between two paralogs, PSD-95 and synapse-associated protein 102 (SAP102) to regulate synaptic AMPARs. During synaptic development, the SAP102 protein levels normally plateau but double if PSD-95 expression is prevented during synaptogenesis. For an autonomous function of PSD-95 in regulating synaptic AMPARs, in addition to the previously demonstrated N-terminal multimerization and the first two PDZ (PSD-95, Dlg1, zona occludens-1) domains, the PDZ3 and guanylate kinase domains were required. The Src homology 3 domain was dispensable for the PSD-95-autonomous regulation of basal synaptic transmission. However, it mediated the functional interaction with SAP102 of PSD-95 mutants to enhance AMPARs. These results depict a protein domain-based multifunctional aspect of PSD-95 in regulating excitatory synaptic transmission and unveil a novel form of domain-based interplay between signaling scaffolds of the DLG-MAGUK family.

Adenylyl Cyclase Anchoring by a Kinase Anchor Protein AKAP5 (AKAP79/150) Is Important for Postsynaptic β-Adrenergic Signaling

Recent evidence indicates that the A kinase anchor protein AKAP5 (AKAP79/150) interacts not only with PKA but also with various adenylyl cyclase (AC) isoforms. However, the physiological relevance of AC-AKAP5 binding is largely unexplored. We now show that postsynaptic targeting of AC by AKAP5 is important for phosphorylation of the AMPA-type glutamate receptor subunit GluA1 on Ser-845 by PKA and for synaptic plasticity. Phosphorylation of GluA1 on Ser-845 is strongly reduced (by 70%) under basal conditions in AKAP5 KO mice but not at all in D36 mice, in which the PKA binding site of AKAP5 (i.e. the C-terminal 36 residues) has been deleted without affecting AC association with GluA1. The increase in Ser-845 phosphorylation upon β-adrenergic stimulation is much more severely impaired in AKAP5 KO than in D36 mice. In parallel, long term potentiation induced by a 5-Hz/180-s tetanus, which mimics the endogenous θ-rhythm and depends on β-adrenergic stimulation, is only modestly affected in acute forebrain slices from D36 mice but completely abrogated in AKAP5 KO mice. Accordingly, anchoring of not only PKA but also AC by AKAP5 is important for regulation of postsynaptic functions and specifically AMPA receptor activity.

The N and C Termini of ZO-1 Are Surrounded by Distinct Proteins and Functional Protein Networks

Biotin ligase tagging with ZO-1 was applied to identify a more complete tight junction proteome. Identical but also different proteins and functional networks were identified near the N and C ends of ZO-1. The ends of ZO-1 are embedded in different functional subcompartments of the tight junction. Biotin tagging with ZO-1 expands the tight junction proteome and defines subcompartments of the junction. The proteins and functional protein networks of the tight junction remain incompletely defined. Among the currently known proteins are barrier-forming proteins like occludin and the claudin family; scaffolding proteins like ZO-1; and some cytoskeletal, signaling, and cell polarity proteins. To define a more complete list of proteins and infer their functional implications, we identified the proteins that are within molecular dimensions of ZO-1 by fusing biotin ligase to either its N or C terminus, expressing these fusion proteins in Madin-Darby canine kidney epithelial cells, and purifying and identifying the resulting biotinylated proteins by mass spectrometry. Of a predicted proteome of ∼9000, we identified more than 400 proteins tagged by biotin ligase fused to ZO-1, with both identical and distinct proteins near the N- and C-terminal ends. Those proximal to the N terminus were enriched in transmembrane tight junction proteins, and those proximal to the C terminus were enriched in cytoskeletal proteins. We also identified many unexpected but easily rationalized proteins and verified partial colocalization of three of these proteins with ZO-1 as examples. In addition, functional networks of interacting proteins were tagged, such as the basolateral but not apical polarity network. These results provide a rich inventory of proteins and potential novel insights into functions and protein networks that should catalyze further understanding of tight junction biology. Unexpectedly, the technique demonstrates high spatial resolution, which could be generally applied to defining other subcellular protein compartmentalization.

Crystal structure of the guanylate kinase domain from discs large homolog 1 (DLG1/SAP97)

Discs large homolog 1 (DLG1/SAP97) is involved in the development and regulation of neuronal and immunological synapses. DLG1 is a member of the membrane associated guanylate kinase (MAGUK) family of proteins, which function as molecular scaffolds. The C-terminal guanylate kinase (GK) domain of DLG1 binds peptides with a phosphorylated serine residue. In this study, we solved the crystal structure of the GK domain of human DLG1. The C-terminal tail of DLG1 is bound to the peptide-binding site of an adjacent symmetry-related DLG1 GK molecule. The binding direction of the C-terminal tail to the peptide-binding site is opposite to that of the phosphorylated LGN peptide in complex with the rat DLG1 GK domain. The C-terminal tail forms a 310 helix, which is also different from the conformation of the phosphorylated LGN peptide. Nevertheless, the side chain interactions of the C-terminal tail with the DLG1 GK domain are similar to those of the phosphorylated LGN peptide.

Role of TARP interaction in S-SCAM-mediated regulation of AMPA receptors

Scaffolding proteins are involved in the incorporation, anchoring, maintenance, and removal of AMPA receptors (AMPARs) at synapses, either through a direct interaction with AMPARs or via indirect association through auxiliary subunits of transmembrane AMPAR regulatory proteins (TARPs). Synaptic scaffolding molecule (S-SCAM) is a newly characterized member of the scaffolding proteins critical for the regulation and maintenance of AMPAR levels at synapses, and directly binds to TARPs through a PDZ interaction. However, the functional significance of S-SCAM–TARP interaction in the regulation of AMPARs has not been tested. Here we show that overexpression of the C-terminal peptide of TARP-γ2 fused to EGFP abolished the S-SCAM-mediated enhancement of surface GluA2 expression. Conversely, the deletion of the PDZ-5 domain of S-SCAM that binds TARPs greatly attenuated the S-SCAM-induced increase of surface GluA2 expression. In contrast, the deletion of the guanylate kinase domain of S-SCAM did not show a significant effect on the regulation of AMPARs. Together, these results suggest that S-SCAM is regulating AMPARs through TARPs.

Crystal Structures of the Scaffolding Protein LGN Reveal the General Mechanism by Which GoLoco Binding Motifs Inhibit the Release of GDP from Gαi

GoLoco (GL) motif-containing proteins regulate G protein signaling by binding to Gα subunit and acting as guanine nucleotide dissociation inhibitors. GLs of LGN are also known to bind the GDP form of Gα(i/o) during asymmetric cell division. Here, we show that the C-terminal GL domain of LGN binds four molecules of Gα(i)·GDP. The crystal structures of Gα(i)·GDP in complex with LGN GL3 and GL4, respectively, reveal distinct GL/Gα(i) interaction features when compared with the only high resolution structure known with GL/Gα(i) interaction between RGS14 and Gα(i1.) Only a few residues C-terminal to the conserved GL sequence are required for LGN GLs to bind to Gα(i)·GDP. A highly conserved "double Arg finger" sequence (RΨ(D/E)(D/E)QR) is responsible for LGN GL to bind to GDP bound to Gα(i). Together with the sequence alignment, we suggest that the LGN GL/Gα(i) interaction represents a general binding mode between GL motifs and Gα(i). We also show that LGN GLs are potent guanine nucleotide dissociation inhibitors.

A Short Polybasic Segment between the Two Conserved Domains of the β2a-Subunit Modulates the Rate of Inactivation of R-type Calcium Channel

Besides opening and closing, high voltage-activated calcium channels transit to a nonconducting inactivated state from which they do not re-open unless the plasma membrane is repolarized. Inactivation is critical for temporal regulation of intracellular calcium signaling and prevention of a deleterious rise in calcium concentration. R-type high voltage-activated channels inactivate fully in a few hundred milliseconds when expressed alone. However, when co-expressed with a particular β-subunit isoform, β(2a), inactivation is partial and develops in several seconds. Palmitoylation of a unique di-cysteine motif at the N terminus anchors β(2a) to the plasma membrane. The current view is that membrane-anchored β(2a) immobilizes the channel inactivation machinery and confers slow inactivation phenotype. β-Subunits contain one Src homology 3 and one guanylate kinase domain, flanked by variable regions with unknown structures. Here, we identified a short polybasic segment at the boundary of the guanylate kinase domain that slows down channel inactivation without relocating a palmitoylation-deficient β(2a) to the plasma membrane. Substitution of the positively charged residues within this segment by alanine abolishes its slow inactivation-conferring phenotype. The linker upstream from the polybasic segment, but not the N- and C-terminal variable regions, masks the effect of this determinant. These results reveal a novel mechanism for inhibiting voltage-dependent inactivation of R-type calcium channels by the β(2a)-subunit that might involve electrostatic interactions with an unknown target on the channel's inactivation machinery or its modulatory components. They also suggest that intralinker interactions occlude the action of the polybasic segment and that its functional availability is regulated by the palmitoylated state of the β(2a)-subunit.

A Quartet of Leucine Residues in the Guanylate Kinase Domain of CaVβ Determines the Plasma Membrane Density of the CaV2.3 Channel

Ca(V)β subunits are formed by a Src homology 3 domain and a guanylate kinase-like (GK) domain connected through a variable HOOK domain. Complete deletion of the Src homology 3 domain (75 residues) as well as deletion of the HOOK domain (47 residues) did not alter plasma membrane density of Ca(V)2.3 nor its typical activation gating. In contrast, six-residue deletions in the GK domain disrupted cell surface trafficking and functional expression of Ca(V)2.3. Mutations of residues known to carry nanomolar affinity binding in the GK domain of Ca(V)β (P175A, P179A, M195A, M196A, K198A, S295A, R302G, R307A, E339G, N340G, and A345G) did not significantly alter cell surface targeting or gating modulation of Ca(V)2.3. Nonetheless, mutations of a quartet of leucine residues (either single or multiple mutants) in the α3, α6, β10, and α9 regions of the GK domain were found to significantly impair cell surface density of Ca(V)2.3 channels. Furthermore, the normalized protein density of Ca(V)2.3 was nearly abolished with the quadruple Ca(V)β3 Leu mutant L200G/L303G/L337G/L342G. Altogether, our observations suggest that the four leucine residues in Ca(V)β3 form a hydrophobic pocket surrounding key residues in the α-interacting domain of Ca(V)2.3. This interaction appears to play an essential role in conferring Ca(V)β-induced modulation of the protein density of Ca(V)α1 subunits in Ca(V)2 channels.

Cacnb4 directly couples electrical activity to gene expression, a process defective in juvenile epilepsy

Calcium current through voltage-gated calcium channels (VGCC) controls gene expression. Here, we describe a novel signalling pathway in which the VGCC Cacnb4 subunit directly couples neuronal excitability to transcription. Electrical activity induces Cacnb4 association to Ppp2r5d, a regulatory subunit of PP2A phosphatase, followed by (i) nuclear translocation of Cacnb4/Ppp2r5d/PP2A, (ii) association with the tyrosine hydroxylase (TH) gene promoter through the nuclear transcription factor thyroid hormone receptor alpha (TRα), and (iii) histone binding through association of Cacnb4 with HP1γ concomitantly with Ser(10) histone H3 dephosphorylation by PP2A. This signalling cascade leads to TH gene repression by Cacnb4 and is controlled by the state of interaction between the SH3 and guanylate kinase (GK) modules of Cacnb4. The human R482X CACNB4 mutation, responsible for a form of juvenile myoclonic epilepsy, prevents association with Ppp2r5 and nuclear targeting of the complex by altering Cacnb4 conformation. These findings demonstrate that an intact VGCC subunit acts as a repressor recruiting platform to control neuronal gene expression.

Ahnak1 interaction is affected by phosphorylation of Ser-296 on Cavβ2

Ahnak1 has been implicated in protein kinase A (PKA)-mediated control of cardiac L-type Ca2+ channels (Cav1.2) through its interaction with the Cavβ2 regulatory channel subunit. Here we corroborate this functional linkage by immunocytochemistry on isolated cardiomyocytes showing co-localization of ahnak1 and Cavβ2 in the T-tubule system. In previous studies Cavβ2 attachment sites which impacted the channel’s PKA regulation have been located to ahnak1’s proximal C-terminus (ahnak14889–5535, ahnak15462–5535). In this study, we mapped the ahnak1-interacting regions in Cavβ2 and investigated whether Cavβ2 phosphorylation affects its binding behavior. In vitro binding assays with Cavβ2 truncation mutants and ahnak14889–5535 revealed that the core region of Cavβ2 consisting of Src-homology 3 (SH3), HOOK, and guanylate kinase (GK) domains was important for ahnak1 interaction while the C- and N-terminal regions were dispensable. Furthermore, Ser-296 in the GK domain of Cavβ2 was identified as novel PKA phosphorylation site by mass spectrometry. Surface plasmon resonance (SPR) binding analysis showed that Ser-296 phosphorylation did not affect the high affinity interaction (KD≈35nM) between Cavβ2 and the α1C I–II linker, but affected ahnak1 interaction in a complex manner. SPR experiments with ahnak15462–5535 revealed that PKA phosphorylation of Cavβ2 significantly increased the binding affinity and, in parallel, it reduced the binding capacity. Intriguingly, the phosphorylation mimic substitution Glu-296 fully reproduced both effects, increased the affinity by ≈2.4-fold and reduced the capacity by ≈60%. Our results are indicative for the release of a population of low affinity interaction sites following Cavβ2 phosphorylation on Ser-296. We propose that this phosphorylation event is one mechanism underlying ahnak1´s modulator function on Cav1.2 channel activity.

Structural Modeling of Full Length Maguk Scaffold Proteins using Single Molecule FRET Restraints

By conjoining protein interaction domains, scaffold proteins form a framework to organize signaling. Synaptic MAGUK scaffold proteins contain three PDZ domains, an SH3 domain and an inactive Guanylate kinase (GK) domain, which are connected by flexible linkers. We used single molecule fluorescence to probe the structure of MAGUK proteins in their isolated “ground” state. We found that the five domains partition into two independent units with relatively fixed structures. From these findings, it is unclear how allosteric coupling between the PDZ domains would be possible.

A Short Basic Segment within a Non-Conserved Region of the Beta-Subunit Modulates the Rate of Inactivation of the R-Type Calcium Channel

Besides opening and closing, high-voltage activated (HVA) calcium channels transit to an inactivated state from which they do not re-open unless the plasma membrane is repolarized. This process is critical for the temporal regulation of intracellular calcium signaling. CaV2.3, a particular class of HVA channels supporting R-type calcium currents, inactivates fully in few hundred milliseconds when expressed alone but when co-expressed with the palmitoylated form of the regulatory β-subunit (CaVβ2a), this process is slowed several fold and is incomplete. The widely accepted view is that membrane-anchoring of the CaVβ2a immobilizes the channel inactivation machinery. Some evidence suggests that an additional structural determinant may be coded in the linker peptide that joins the two highly conserved domains composing CaVβ but whether these determinants target the subunit to the membrane has not been shown. Here we indentify a short positively charged segment lying at the boundary of the guanylate kinase domain of CaVβ2a that slows down channel inactivation without relocating the protein to the plasma membrane. Deletion analysis demonstrates that while neither the N-terminal nor the C-terminal variable region of the protein affects the regulatory effect of the basic segment the presence of the remaining residues within the linker does. These results demonstrate that membrane anchoring is not the only factor modulating inactivation rate CaV2.3 calcium channels and suggest the presence of intralinker interactions or posttranslational modifications that counteract the effect of this segment.

Structural Modeling of PSD-95 and other MAGUKs using Single-Molecule FRET

In order for information to be correctly propagated between cells, it is necessary for both the sending and receiving cells to have organized molecular machinery so that the transmitted signals can be accurately received and processed. By conjoining protein interaction domains, multidomain scaffold proteins form a framework for maintaining and modulating these junctional communications. Scaffold proteins have been traditionally conceptualized as “beads on a string” with unstructured linkers allowing each domain to be independently oriented, but recent data on multidomain supramodules suggests that the fundamental functional units of scaffold proteins may be larger multidomain complexes. Membrane-associated guanylate kinase (MAGUK) proteins are multidomain scaffold proteins that function at cellular junctions; most importantly at epithelial tight junctions and within the postsynaptic densities of neurons. MAGUK proteins contain both PSD-95/Dlg/ZO-1 (PDZ) and Src Homology (SH3) domains, as well as an enzymatically inactive guanylate kinase domain. While the high-resolution structures of the isolated domains of the canonical MAGUK, PSD-95, have been solved, the organization of these domains remains unknown. In combination with rigid body docking and all-atom molecular dynamics simulations, single-molecule FRET measurements between each of five domains allowed for the determination of the domain positioning in full-length PSD-95. The “ground” state configuration of PSD-95 was found to contain two stable multidomain subunits that while connected by a flexible linker do not appear to interact. A comparison of measurements within each of these partitions with homologous measurements in other MAGUKs showed that domain orientations can vary among this family of scaffold proteins. These findings represent the first unambiguous assignment of domain positioning in a full-length scaffold protein and provide insights into potential allosteric coupling between domains in MAGUK proteins.

Guanylate kinase domains of the MAGUK family scaffold proteins as specific phospho-protein-binding modules

Membrane-associated guanylate kinases (MAGUKs) are a large family of scaffold proteins that play essential roles in tissue developments, cell-cell communications, cell polarity control, and cellular signal transductions. Despite extensive studies over the past two decades, the functions of the signature guanylate kinase domain (GK) of MAGUKs are poorly understood. Here we show that the GK domain of DLG1/SAP97 binds to asymmetric cell division regulatory protein LGN in a phosphorylation-dependent manner. The structure of the DLG1 SH3-GK tandem in complex with a phospho-LGN peptide reveals that the GMP-binding site of GK has evolved into a specific pSer/pThr-binding pocket. Residues both N- and C-terminal to the pSer are also critical for the specific binding of the phospho-LGN peptide to GK. We further demonstrate that the previously reported GK domain-mediated interactions of DLGs with other targets, such as GKAP/DLGAP1/SAPAP1 and SPAR, are also phosphorylation dependent. Finally, we provide evidence that other MAGUK GKs also function as phospho-peptide-binding modules. The discovery of the phosphorylation-dependent MAGUK GK/target interactions indicates that MAGUK scaffold-mediated signalling complex organizations are dynamically regulated.

The Structure of the PDZ3-SH3-GuK Tandem of ZO-1 Protein Suggests a Supramodular Organization of the Membrane-associated Guanylate Kinase (MAGUK) Family Scaffold Protein Core

Membrane-associated guanylate kinases (MAGUKs) are a large family of scaffold proteins that play essential roles in tethering membrane receptors, adhesion molecules, and macromolecular signaling complexes for tissue developments, cell-cell communications, and intracellular signal transductions. The defining feature of the MAGUK family scaffolds is that each member contains a conserved core consisting of a PSD-95/Dlg/ZO-1 (PDZ) domain, an Src homology 3 (SH3) domain, and a catalytically inactive guanylate kinase (GuK) domain arranged in tandem, although the structural features and functional implications of the PDZ-SH3-GuK tandem arrangement are unclear. The structure of the ZO-1 PDZ3-SH3-GuK tandem solved in this study reveals that the PDZ domain directly interacts with the SH3-GuK module, forming a structural supramodule with distinct target binding properties with respect to the isolated domains. Structure-based sequence analysis suggests that the PDZ-SH3-GuK tandems of other members of the MAGUK family also form supramodules.

Consensus Substrate Sequence for Protein-tyrosine Phosphatase Receptor Type Z

Protein-tyrosine phosphatase receptor type Z (Ptprz) has multiple substrate proteins, including G protein-coupled receptor kinase-interactor 1 (Git1), membrane-associated guanylate kinase, WW and PDZ domain-containing 1 (Magi1), and GTPase-activating protein for Rho GTPase (p190RhoGAP). We have identified a dephosphorylation site at Tyr-1105 of p190RhoGAP; however, the structural determinants employed for substrate recognition of Ptprz have not been fully defined. In the present study, we revealed that Ptprz selectively dephosphorylates Git1 at Tyr-554, and Magi1 at Tyr-373 and Tyr-858 by in vitro and cell-based assays. Of note, the dephosphorylation of the Magi1 Tyr-858 site required PDZ domain-mediated interaction between Magi1 and Ptprz in the cellular context. Alignment of the primary sequences surrounding the target phosphotyrosine residue in these three substrates showed considerable similarity, suggesting a consensus motif for recognition by Ptprz. We then estimated the contribution of surrounding individual amino acid side chains to the catalytic efficiency by using fluorescent peptides based on the Git1 Tyr-554 sequence in vitro. The typical substrate motif for the catalytic domain of Ptprz was deduced to be Glu/Asp-Glu/Asp-Glu/Asp-Xaa-Ile/Val-Tyr(P)-Xaa (Xaa is not an acidic residue). Intriguingly, a G854D substitution of the Magi1 Tyr-858 site matching better to the motif sequence turned this site to be susceptible to dephosphorylation by Ptprz independent of the PDZ domain-mediated interaction in cells. Furthermore, we found by database screening that the substrate motif is present in several proteins, including paxillin at Tyr-118, its major phosphorylation site. Expectedly, we verified that Ptprz efficiently dephosphorylates paxillin at this site in cells. Our study thus provides key insights into the molecular basis for the substrate recognition of Ptprz.

Phosphorylation of a PDZ Domain Extension Modulates Binding Affinity and Interdomain Interactions in Postsynaptic Density-95 (PSD-95) Protein, a Membrane-associated Guanylate Kinase (MAGUK)

Postsynaptic density-95 is a multidomain scaffolding protein that recruits glutamate receptors to postsynaptic sites and facilitates signal processing and connection to the cytoskeleton. It is the leading member of the membrane-associated guanylate kinase family of proteins, which are defined by the PSD-95/Discs large/ZO-1 (PDZ)-Src homology 3 (SH3)-guanylate kinase domain sequence. We used NMR to show that phosphorylation of conserved tyrosine 397, which occurs in vivo and is located in an atypical helical extension (α3), initiates a rapid equilibrium of docked and undocked conformations. Undocking reduced ligand binding affinity allosterically and weakened the interaction of PDZ3 with SH3 even though these domains are separated by a ~25-residue linker. Additional phosphorylation at two linker sites further disrupted the interaction, implicating α3 and the linker in tuning interdomain communication. These experiments revealed a novel mode of regulation by a detachable PDZ element and offer a first glimpse at the dynamic interaction of PDZ and SH3-guanylate kinase domains in membrane-associated guanylate kinases.

Downregulation of MAGI1 Associates with Poor Prognosis of Hepatocellular Carcinoma

ABSTRACTObjective: MAGI1 (membrane-associated guanylate kinase, WW and PDZ domain containing 1) plays an important role in stabilization of adherens junctions and suppression of invasiveness and metastasis. However, its expression and clinical revelance in hepatocellular carcinoma (HCC) are still unknown. So, this study was performed to detect the expression and clinical significance of MAGI1 in human HCC. Methods: The mRNA and protein expression levels were examined by using reverse transcription-PCR (RT-PCR) and Western blotting in 31 paired HCC and paracarcinomatous liver tissues (PCLT). Furthermore, samples of 104 HCC patients were determined immunohistochemically for MAGI1 expression and the correlation of MAGI1 levels with prognosis was analyzed. Results: The expression levels of MAGI1 were significantly downregulated in HCCs than those in PCLTs (p < .01). Importantly, the decreased expression of MAGI1 correlated with multiple tumor nodules (p < .05), absence of capsular formation (p < .05), worse Edmondson–Steiner grade (p < .05), vein invasion (p < .01), shortened median overall survival time (25 versus 42 months; p = .013), and disease-free survival time (20 versus 40 months; p = .018) of HCC. Multivariable Cox regression analysis revealed that MAGI1 expression level was an independent factor for prognosis (p = .038). Conclusions: MAGI1 expression is decreased in HCC, which correlates with poor prognosis, suggesting MAGI1 as a novel prognostic marker for HCC.

MAGI1 inhibits cancer cell migration and invasion of hepatocellular carcinoma via regulating PTEN.

To explore the biological function and molecular mechanism of membrane associated guanylate kinase, WW and PDZ domain containing 1 (MAGI1) in hepatocellular carcinoma. HepG2(MAGI1) stable cell line was constructed by transfecting HepG2 cells with pcDNA3.1-MAGI1 plasmid. Wound healing and invasion assay were performed to compare the migration and invasion ability of HepG2(MAGI1) and HepG2 cells. Furthermore, the expression of MAGI1 and phosphatase and tensin homolog deleted on chromosome ten (PTEN) was also examined by Western blot and the relationship was analyzed. The wound healing assay showed that the closure of HepG2(MAGI1) cells was significantly slower than that of HepG2 cells [(90 ± 10)% vs. (50 ± 15)%, P<0.05], and the invasion assay showed that the number of HepG2(MAGI1) cells that passed through the matrigel was fewer than HepG2 cells (68 ± 18 vs. 150 ± 30, P<0.05). The protein expression level of PTEN was significantly elevated in HepG2(MAGI1) cells compared with HepG2 cells (1.40 ± 0.32 vs. 0.28 ± 0.15, P<0.05). MAGI1 and PTEN protein expression levels were positively correlated (r=0.913, P<0.01). MAGI1 may inhibit the cancer cell migration and invasion of hepatocellular carcinoma via regulating PTEN.