The small molecule inhibitor NAV-2729 has a complex target profile including multiple ADP-ribosylation factor regulatory proteins.

The ADP-ribosylation factor (Arf) GTPases and their regulatory proteins are implicated in cancer progression. NAV-2729 was previously identified as a specific inhibitor of Arf6 that reduced progression of uveal melanoma in an orthotopic xenograft. Here, our goal was to assess the inhibitory effects of NAV-2729 on the proliferation of additional cell types. We found NAV-2729 inhibited proliferation of multiple cell lines, but Arf6 expression did not correlate with NAV-2729 sensitivity, and knockdown of Arf6 affected neither cell viability nor sensitivity to NAV-2729. Furthermore, binding to native Arf6 was not detected; however, we determined that NAV-2729 inhibited both Arf exchange factors and Arf GTPase-activating proteins. ASAP1, a GTPase-activating protein linked to cancer progression, was further investigated. We demonstrated that NAV-2729 bound to the PH domain of ASAP1 and changed ASAP1 cellular distribution. However, ASAP1 knockdown did not fully recapitulate the cytoskeletal effects of NAV-2729 nor affect cell proliferation. Finally, our screens identified 48 other possible targets of NAV-2729. These results illustrate the complexities of defining targets of small molecules and identify NAV-2729 as a model PH domain-binding inhibitor.

Dual-layer spectral detector CT arthrography of the shoulder: assessment of image quality and value in differentiating calcium from iodine.

BACKGROUND: Dual-layer spectral detector computed tomography (DLCT) may potentially improve CT arthrography through enhanced image quality and analysis of the chemical composition of tissue. PURPOSE: To evaluate the image quality of monoenergetic reconstructions from DLCT arthrography of the shoulder and assess the additional diagnostic value in differentiating calcium from iodine. MATERIAL AND METHODS: Images from consecutive shoulder DLCT arthrography examinations performed between December 2016 and February 2018 were retrospectively reviewed for hyperattenuating lesions within the labrum and tendons. The mean attenuation of the target lesion, noise, contrast-to-noise ratio (CNR), and signal-to-noise ratio (SNR) of the virtual monoenergetic images obtained at 40-200 keV were compared with conventional 140-kVp images. Two evaluators independently classified each target lesion as contrast media or calcification, without and with DLCT spectral data. Receiver operating curve (ROC) analysis was performed to assess the diagnostic performance of shoulder DLCT arthrography, without and with the aid of spectral data. RESULTS: The study included 20 target lesions (18 DLCT arthrography examinations of 17 patients). The SNRs of the monoenergetic images at 40-60 keV were significantly higher than those of conventional images (P < 0.05). The CNRs of the monoenergetic images at 40-70 keV were significantly higher than those of conventional images (P < 0.001). The ability to differentiate calcium from iodine, without and with DLCT spectral data, did not significantly differ (P = 0.441 and P = 0.257 for reviewers 1 and 2, respectively). CONCLUSION: DLCT had no additive value in differentiating calcium from iodine in small, hyperattenuating lesions in the labrum and tendons.

SOCS3 Attenuates Dexamethasone-Induced M2 Polarization by Down-Regulation of GILZ via ROS- and p38 MAPK-Dependent Pathways.

Suppressors of cytokine signaling (SOCS) have emerged as potential regulators of macrophage function. We have investigated mechanisms of SOCS3 action on type 2 macrophage (M2) differentiation induced by glucocorticoid using human monocytic cell lines and mouse bone marrow-derived macrophages. Treatment of THP1 monocytic cells with dexamethasone (Dex) induced ROS generation and M2 polarization promoting IL-10 and TGF-ß production, while suppressing IL-1ß, TNF-α and IL-6 production. SOCS3 over-expression reduced, whereas SOCS3 ablation enhanced IL-10 and TGF-ß induction with concomitant regulation of ROS. As a mediator of M2 differentiation, glucocorticoid-induced leucine zipper (GILZ) was down-regulated by SOCS3 and up-regulated by shSOCS3. The induction of GILZ and IL-10 by Dex was dependent on ROS and p38 MAPK activity. Importantly, GILZ ablation led to the inhibition of ROS generation and anti-inflammatory cytokine induction by Dex. Moreover, GILZ knock-down negated the up-regulation of IL-10 production induced by shSOCS3 transduction. Our data suggest that SOCS3 targets ROS- and p38-dependent GILZ expression to suppress Dex-induced M2 polarization.

ERK phosphorylation is dependent on cell adhesion in a subset of pediatric sarcoma cell lines.

Osteosarcoma (OS) and Pax-Foxo1 fusion negative rhabdomyosarcoma (FN-RMS) are pediatric sarcomas with poor prognoses in patients with advanced disease. In both malignancies, an actin binding protein has been linked to poor prognosis. Integrin adhesion complexes (IACs) are closely coupled to actin networks and IAC-mediated signaling has been implicated in the progression of carcinomas. However, the relationship of IACs and actin cytoskeleton remodeling with cell signaling is understudied in pediatric sarcomas. Here, we tested the hypothesis that IAC dynamics affect ERK activation in OS and FN-RMS cell lines. Adhesion dependence of ERK activation differed among the OS and FN-RMS cells examined. In the OS cell lines, adhesion did not have a consistent effect on phospho-ERK (pERK). ERK phosphorylation in response to fetal calf serum or 1 ng/ml EGF was nearly as efficient in OS cell lines and one FN-RMS cell line in suspension as cells adherent to poly-l-lysine (PL) or fibronectin (FN). By contrast, adhesion to plastic, PL or FN increased ERK phosphorylation and was greater than additive with a 15 min exposure to 1 ng/ml EGF in three FN-RMS cell lines. Increases in pERK were partly dependent on FAK and PAK1/2 but independent of IAC maturation. As far as we are aware, this examination of adhesion-dependent signaling is the first in pediatric sarcomas and has led to the discovery of differences from the prevailing paradigms and differences in the degree of coupling between components in the signaling pathways among the cell lines.

A lysine-rich cluster in the N-BAR domain of ARF GTPase-activating protein ASAP1 is necessary for binding and bundling actin filaments.

Actin filament maintenance is critical for both normal cell homeostasis and events associated with malignant transformation. The ADP-ribosylation factor GTPase-activating protein ASAP1 regulates the dynamics of filamentous actin-based structures, including stress fibers, focal adhesions, and circular dorsal ruffles. Here, we have examined the molecular basis for ASAP1 association with actin. Using a combination of structural modeling, mutagenesis, and in vitro and cell-based assays, we identify a putative-binding interface between the N-Bin-Amphiphysin-Rvs (BAR) domain of ASAP1 and actin filaments. We found that neutralization of charges and charge reversal at positions 75, 76, and 79 of ASAP1 reduced the binding of ASAP1 BAR-pleckstrin homology tandem to actin filaments and abrogated actin bundle formation in vitro. In addition, overexpression of actin-binding defective ASAP1 BAR-pleckstrin homology [K75, K76, K79] mutants prevented cellular actin remodeling in U2OS cells. Exogenous expression of [K75E, K76E, K79E] mutant of full-length ASAP1 did not rescue the reduction of cellular actin fibers consequent to knockdown of endogenous ASAP1. Taken together, our results support the hypothesis that the lysine-rich cluster in the N-BAR domain of ASAP1 is important for regulating actin filament organization.

Sonographic Appearance of Steatocystoma: An Analysis of 14 Pathologically Confirmed Lesions.

Purpose: To evaluate the ultrasonographic characteristics of steatocystomas focusing on the features that aid in differentiating them from epidermal inclusion cysts and lipomas. Materials and Methods: The ultrasonographic findings of 14 histologically proven steatocystomas in 10 patients were retrospectively reviewed. The following features were assessed: the layer of involvement, shape, margin, echogenicity, posterior acoustic features, and the presence of a visible wall or intralesional striations. The findings were compared with those of subcutaneous lipomas and epidermal inclusion cysts to identify those findings that aid in the differential diagnosis of steatocystomas. Results: The majority of steatocystomas appeared as a subcutaneous mass (n = 6, 42.9%) or a mass involving both the dermal and subcutaneous layers (n = 6, 42.9%). Steatocystomas exhibited a well-defined smooth margin (n = 12, 85.7%) and homogeneous echogenicity (n = 9, 64.3%), and showed no specific posterior acoustic features (n = 9, 64.3%). The most important features that differentiated steatocystomas from epidermal inclusion cysts were a homogeneous internal echotexture (p = 0.009) and absent or less prominent posterior acoustic enhancement (p < 0.001). The features that distinguished steatocystomas from lipomas were the margin (p < 0.001), echogenicity (p = 0.034), internal echotexture (p = 0.004), and the absence of intralesional striations (p < 0.001). Conclusion: Steatocystomas appeared as well-defined homogeneous masses with mild or absent posterior acoustic enhancement.

Anti-inflammatory mechanisms of suppressors of cytokine signaling target ROS via NRF-2/thioredoxin induction and inflammasome activation in macrophages.

Suppressors of cytokine signaling (SOCS) exhibit diverse antiinflammatory effects. Since ROS acts as a critical mediator of inflammation, we have investigated the anti-inflammatory mechanisms of SOCS via ROS regulation in monocytic/macrophagic cells. Using PMA-differentiated monocytic cell lines and primary BMDMs transduced with SOCS1 or shSOCS1, the LPS/TLR4-induced inflammatory signaling was investigated by analyzing the levels of intracellular ROS, antioxidant factors, inflammasome activation, and pro-inflammatory cytokines. The levels of LPS-induced ROS and the production of pro-inflammatory cytokines were notably down-regulated by SOCS1 and up-regulated by shSOCS1 in an NAC-sensitive manner. SOCS1 up-regulated an ROS-scavenging protein, thioredoxin, via enhanced expression and binding of NRF-2 to the thioredoxin promoter. SOCS3 exhibited similar effects on NRF-2/thioredoxin induction, and ROS downregulation, resulting in the suppression of inflammatory cytokines. Notably thioredoxin ablation promoted NLRP3 inflammasome activation and restored the SOCS1-mediated inhibition of ROS and cytokine synthesis induced by LPS. The results demonstrate that the anti-inflammatory mechanisms of SOCS1 and SOCS3 in macrophages are mediated via NRF-2-mediated thioredoxin upregulation resulting in the downregulation of ROS signal. Thus, our study supports the anti-oxidant role of SOCS1 and SOCS3 in the exquisite regulation of macrophage activation under oxidative stress. [BMB Reports 2020; 53(12): 640-645].

ARAP2 signals through Arf6 and Rac1 to control focal adhesion morphology.

Focal adhesions (FAs) are dynamic structures that connect the actin cytoskeleton with the extracellular matrix. At least six ADP-ribosylation factor (Arf) GTPase-activating proteins (GAPs), including ARAP2 (an Arf6 GAP), are implicated in regulation of FAs but the mechanisms for most are not well defined. Although Rac1 has been reported to function downstream of Arf6 to control membrane ruffling and cell migration, this pathway has not been directly examined as a regulator of FAs. Here we test the hypothesis that ARAP2 promotes the growth of FAs by converting Arf6·GTP to Arf6·GDP thereby preventing the activation of the Rho family GTP-binding protein Rac1. Reduced expression of ARAP2 decreased the number and size of FAs in cells and increased cellular Arf6·GTP and Rac1·GTP levels. Overexpression of ARAP2 had the opposite effects. The effects of ARAP2 on FAs and Rac1 were dependent on a functional ArfGAP domain. Constitutively active Arf6 affected FAs in the same way as did reduced ARAP2 expression and dominant negative mutants of Arf6 and Rac1 reversed the effect of reduced ARAP2 expression. However, neither dominant negative Arf6 nor Rac1 had the same effect as ARAP2 overexpression. We conclude that changes in Arf6 and Rac1 activities are necessary but not sufficient for ARAP2 to promote the growth of FAs and we speculate that ARAP2 has additional functions that are effector in nature to promote or stabilize FAs.

ARAP1 association with CIN85 affects epidermal growth factor receptor endocytic trafficking.

BACKGROUND INFORMATION: ARAP1 is an Arf (ADP-ribosylation factor)-directed GAP (GTPase-activating protein) that inhibits the trafficking of EGFR (epidermal growth factor receptor) to the early endosome. To further understand the function of ARAP1, we sought to identify proteins that interact with ARAP1. RESULTS: Here we report that ARAP1 associates with the CIN85 (Cbl-interacting protein of 85 kDa). Arg86 and Arg90 of ARAP1 and the SH3 (Src homology 3) domains of CIN85 are necessary for the interaction. We found that a mutant of ARAP1 with reduced affinity for CIN85 does not efficiently rescue the effect of reduced ARAP1 expression on EGFR trafficking to the early endosome. Reduced expression of CIN85 has a similar effect as reduced expression of ARAP1 on traffic of the EGFR. Cbl proteins regulate the endocytic trafficking of the EGFR by mediating ubiquitination of the EGFR. Overexpression of ARAP1 reduced ubiquitination of the EGFR by Cbl and slowed Cbl-dependent degradation of the EGFR. Reduced expression of ARAP1 accelerated degradation of EGFR but did not affect the level of ubiquitination of the receptor that was detected. CONCLUSION: ARAP1 interaction with CIN85 regulates endocytic trafficking of the EGFR and affects ubiquitination of EGFR. We propose a model in which the ARAP1-CIN85 complex drives exit of EGF-EGFR-Cbl complex from a pre-early endosome into a pathway distinct from the early endosome/lysosome pathway.

PTK6 inhibits down-regulation of EGF receptor through phosphorylation of ARAP1.

PTK6 (also known as Brk) is a non-receptor-tyrosine kinase containing SH3, SH2, and catalytic domains, that is expressed in more than 60% of breast carcinomas but not in normal mammary tissues. To analyze PTK6-interacting proteins, we have expressed Flag-tagged PTK6 in HEK293 cells and performed co-immunoprecipitation assays with Flag antibody-conjugated agarose. A 164-kDa protein in the precipitated fraction was identified as ARAP1 (also known as centaurin delta-2) by MALDI-TOF mass analysis. ARAP1 associated with PTK6 in an EGF/EGF receptor (EGFR)-dependent manner. In addition, the SH2 domain of PTK6, particularly the Arg(105) residue that contacts the phosphate group of the tyrosine residue, was essential for the association. Moreover, PTK6 phosphorylated residue Tyr(231) in the N-terminal domain of ARAP1. Expression of ARAP1, but not of the Y231F mutant, inhibited the down-regulation of EGFR in HEK293 cells expressing PTK6. Silencing of endogenous PTK6 expression in breast carcinoma cells decreased EGFR levels. These results demonstrate that PTK6 enhances EGFR signaling by inhibition of EGFR down-regulation through phosphorylation of ARAP1 in breast cancer cells.

A PH domain in the Arf GTPase-activating protein (GAP) ARAP1 binds phosphatidylinositol 3,4,5-trisphosphate and regulates Arf GAP activity independently of recruitment to the plasma membranes.

ARAP1 is a phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3))-dependent Arf GTPase-activating protein (GAP) with five PH domains that regulates endocytic trafficking of the epidermal growth factor receptor (EGFR). Two tandem PH domains are immediately N-terminal of the Arf GAP domain, and one of these fits the consensus sequence for PtdIns(3,4,5)P(3) binding. Here, we tested the hypothesis that PtdIns(3,4,5)P(3)-dependent recruitment mediated by the first PH domain of ARAP1 regulates the in vivo and in vitro function of ARAP1. We found that PH1 of ARAP1 specifically bound to PtdIns(3,4,5)P(3), but with relatively low affinity (approximately 1.6 microm), and the PH domains did not mediate PtdIns(3,4,5)P(3)-dependent recruitment to membranes in cells. However, PtdIns(3,4,5)P(3) binding to the PH domain stimulated GAP activity and was required for in vivo function of ARAP1 as a regulator of endocytic trafficking of the EGFR. Based on these results, we propose a variation on the model for the function of phosphoinositide-binding PH domains. In our model, ARAP1 is recruited to membranes independently of PtdIns(3,4,5)P(3), the subsequent production of which triggers enzymatic activity.

ARAP1 regulates endocytosis of EGFR.

Signaling through the EGF receptor is regulated by endocytosis. ARAP1 is a protein with Arf guanosine triphosphatase-activating protein (GAP) and Rho GAP domains. We investigated the role of ARAP1 in EGF receptor endocytic trafficking. Following EGF treatment of cells, ARAP1 rapidly and transiently associated with the edge of the cell and punctate structures containing Rab5, rabaptin 5 and EGFR but not early embryonic antigen 1 (EEA1). EGF associated with the ARAP1-positive punctate structures prior to EEA1-positive early endosomes. Recruitment of ARAP1 to the punctate structures required active Rab5 and an additional signal from EGFR. Decreasing ARAP1 levels with small interfering RNA accelerated association of EGF with EEA1 endosomes and degradation of EGFR. Phosphorylation of extracellular-signal-regulated kinase (ERK) and c-Jun-amino-terminal kinase (JNK) was diminished and more transient in cells with reduced levels of ARAP1 than in controls. Based on these findings, we propose that ARAP1 regulates the endocytic traffic of EGFR and, consequently, the rate of EGFR signal attenuation.

In vitro and in vivo analysis of neurotrophin-3 activation of Arf6 and Rac-1.

Arf GTP-binding proteins and Rho-family GTPases play key roles in regulating membrane remodeling and cytoskeletal reorganization involved in cell movement. Several studies have implicated neurotrophins and their receptors as upstream activators of these small GTP-binding proteins, however, the mechanisms and the cell type specificity of this neurotrophin activity are still under investigation. Here we describe the rationale and protocols used for the dissection of an NT3 activated pathway that leads to the specific activation of Arf6 and Rac1.

Src-dependent phosphorylation of ASAP1 regulates podosomes.

Invadopodia are Src-induced cellular structures that are thought to mediate tumor invasion. ASAP1, an Arf GTPase-activating protein (GAP) containing Src homology 3 (SH3) and Bin, amphiphysin, and RVS161/167 (BAR) domains, is a substrate of Src that controls invadopodia. We have examined the structural requirements for ASAP1-dependent formation of invadopodia and related structures in NIH 3T3 fibroblasts called podosomes. We found that both predominant splice variants of ASAP1 (ASAP1a and ASAP1b) associated with invadopodia and podosomes. Podosomes were highly dynamic, with rapid turnover of both ASAP1 and actin. Reduction of ASAP1 levels by small interfering RNA blocked formation of invadopodia and podosomes. Podosomes were formed in NIH 3T3 fibroblasts in which endogenous ASAP1 was replaced with either recombinant ASAP1a or ASAP1b. ASAP1 mutants that lacked the Src binding site or GAP activity functioned as well as wild-type ASAP1 in the formation of podosomes. Recombinant ASAP1 lacking the BAR domain, the SH3 domain, or the Src phosphorylation site did not support podosome formation. Based on these results, we conclude that ASAP1 is a critical target of tyrosine kinase signaling involved in the regulation of podosomes and invadopodia and speculate that ASAP1 may function as a coincidence detector of simultaneous protein association through the ASAP1 SH3 domain and phosphorylation by Src.

ARAP2 effects on the actin cytoskeleton are dependent on Arf6-specific GTPase-activating-protein activity and binding to RhoA-GTP.

ARAP2 is a protein that contains both ArfGAP and RhoGAP domains. We found that it is a phosphatidylinositol (3,4,5)-trisphosphate-dependent Arf6 GAP that binds RhoA-GTP but lacks RhoGAP activity. In agreement with the hypothesis that ARAP2 mediates effects of RhoA, endogenous ARAP2 associated with focal adhesions (FAs) and reduction of ARAP2 expression, by RNAi, resulted in fewer FAs and actin stress fibers (SFs). In cells with reduced levels of endogenous ARAP2, FAs and SFs could be restored with wild-type recombinant ARAP2 but not mutants lacking ArfGAP or Rho-binding activity. Constitutively active Arf6 also caused a loss of SFs. The Rho effector ROKalpha was ineffective in restoring FAs. Conversely, overexpression of ARAP2 did not restore SFs in cells treated with a ROK inhibitor but induced punctate accumulations of paxillin. We conclude that ARAP2 is an Arf6GAP that functions downstream of RhoA to regulate focal adhesion dynamics.

A kinase-deficient TrkC receptor isoform activates Arf6-Rac1 signaling through the scaffold protein tamalin.

Neurotrophins play an essential role in mammalian development. Most of their functions have been attributed to activation of the kinase-active Trk receptors and the p75 neurotrophin receptor. Truncated Trk receptor isoforms lacking the kinase domain are abundantly expressed during development and in the adult; however, their function and signaling capacity is largely unknown. We show that the neurotrophin-3 (NT3) TrkCT1-truncated receptor binds to the scaffold protein tamalin in a ligand-dependent manner. Moreover, NT3 initiation of this complex leads to activation of the Rac1 GTPase through adenosine diphosphate-ribosylation factor 6 (Arf6). At the cellular level, NT3 binding to TrkCT1-tamalin induces Arf6 translocation to the membrane, which in turn causes membrane ruffling and the formation of cellular protrusions. Thus, our data identify a new signaling pathway elicited by the kinase-deficient TrkCT1 receptor. Moreover, we establish NT3 as an upstream regulator of Arf6.

Regulation of ASAP1 by phospholipids is dependent on the interface between the PH and Arf GAP domains.

ASAP1 is an Arf GAP with a PH domain immediately N-terminal to the catalytic Arf GAP domain. PH domains are thought to regulate enzymes by binding to specific phosphoinositide lipids in membranes, thereby recruiting the enzyme to a site of action. Here, we have examined the functional relationship between the PH and Arf GAP domains. We found that GAP activity requires the cognate PH domain of ASAP1, leading us to hypothesize that the Arf GAP and PH domains directly interact to form the substrate binding site. This hypothesis was supported by the combined results of protection and hydrodynamic studies. We then examined the role of the PH domain in the regulation of Arf GAP activity. The results of saturation kinetics, limited proteolysis, FRET and fluorescence spectrometry support a model in which regulation of the GAP activity of ASAP1 involves a conformational change coincident with recruitment to a membrane surface, and a second conformational change following the specific binding of phosphatidylinositol 4,5-bisphosphate.

In vitro assays of Arf1 interaction with GGA proteins.

ADP-ribosylation factor 1 (Arf1) is a GTP-binding protein that regulates membrane traffic. This function of Arf1 is, at least in part, mediated by Arf1 x GTP binding to coat proteins such as coatomer, clathrin adaptor protein (AP) complexes 1 and 3, and gamma-adaptin homology-Golgi associated Arf-binding (GGA) proteins. Binding to Arf1 x GTP recruits these coat proteins to membranes, leading to the formation of transport vesicles. Whereas coatomer and the AP complexes are hetero-oligomers, GGAs are single polypeptide chains. Therefore, working with recombinant GGAs is straightforward compared to the other Arf1 effectors. Consequently, the GGAs have been used as a model for studying Arf1 interactions with effectors and as reagents to determine Arf1 x GTP levels in cells. In this chapter, we describe in vitro assays for analysis of GGA interaction with Arf1 x GTP and for determining intracellular Arf1 x GTP levels.

Importance of Gly-13 for the coenzyme binding of human UDP-glucose dehydrogenase.

UDP-glucose dehydrogenase (UGDH) is the unique pathway enzyme furnishing in vertebrates UDP-glucuronate for numerous transferases. In this report, we have identified an NAD(+)-binding site within human UGDH by photoaffinity labeling with a specific probe, [(32)P]nicotinamide 2-azidoadenosine dinucleotide (2N(3) NAD(+)), and cassette mutagenesis. For this work, we have chemically synthesized a 1509-base pair gene encoding human UGDH and expressed it in Escherichia coli as a soluble protein. Photolabel-containing peptides were generated by photolysis followed by tryptic digestion and isolated using the phosphopeptide isolation kit. Photolabeling of these peptides was effectively prevented by the presence of NAD(+) during photolysis, demonstrating a selectivity of the photoprobe for the NAD(+)-binding site. Amino acid sequencing and compositional analysis identified the NAD(+)-binding site of UGDH as the region containing the sequence ICCIGAXYVGGPT, corresponding to Ile-7 through Thr-19 of the amino acid sequence of human UGDH. The unidentified residue, X, can be designated as a photolabeled Gly-13 because the sequences including the glycine residue in question have a complete identity with those of other UGDH species known. The importance of Gly-13 residue in the binding of NAD(+) was further examined with a G13E mutant by cassette mutagenesis. The mutagenesis at Gly-13 had no effects on the expression or stability of the mutant. Enzyme activity of the G13E point mutant was not measurable under normal assay conditions, suggesting an important role for the Gly-13 residue. No incorporation of [(32)P]2N(3)NAD(+) was observed for the G13E mutant. These results indicate that Gly-13 plays an important role for efficient binding of NAD(+) to human UGDH.

Reactive amino acid residues involved in glutamate-binding of human glutamate dehydrogenase isozymes.

In the present study, the cassette mutagenesis at several putative positions (K94, G96, K118, K130, or D172) was performed to examine the residues involved in the glutamate-binding of the human glutamate dehydrogenase isozymes (hGDH1 and hGDH2). None of the mutations tested affected the expression or stability of the proteins. There was dramatic reduction in the catalytic efficiency in mutant proteins at K94, G96, K118, or K130 site, but not at D172 site. The K(M) values for glutamate were 4-10-fold greater for the mutants at K94, G96, or K118 site than for the wild-type hGDH1 and hGDH2, whereas no differences in the K(M) values for NAD(+) were detected between the mutant and wild-type enzymes. For K130Y mutant, the K(M) value for glutamate increased 1.6-fold, whereas the catalytic efficiency (k(cat)/K(M)) showed only 2-3% of the wild-type. Therefore, the decreased catalytic efficiency of the K130 mutant mainly results from the reduced k(cat) value, suggesting a possibility that the K130Y residue may be involved in the catalysis rather than in the glutamate-binding. The D172Y mutant did not show any changes in k(cat) value and K(M) values for glutamate and NAD(+), indicating that D172Y is not directly involved in catalysis and substrates binding of the hGDH isozymes. For sensitivity to ADP activation, only the D172Y mutant showed a reduced sensitivity to ADP activation. The reduction of ADP activation in D172Y mutant was more profoundly observed in hGDH2 than in hGDH1. There were no differences in their sensitivities to GTP inhibition between the wild-type and mutant GDHs at all positions tested. Our results suggest that K94, G96, and K118 residues play an important role, although at different degrees, in the binding of glutamate to hGDH isozymes.