Autophosphorylation In Response Research Articles

Missense variant in insulin receptor (Y1355H) segregates in family with fatty liver disease

A missense variant in the cytoplasmic domain of the insulin receptor (INSR) was identified by exome sequencing in affected members of a four-generation family with fatty liver disease (FLD). The variant (rs766457461, c.4063T>C, p.Y1355H) results in the substitution of histidine for a tyrosine that undergoes autophosphorylation in response to insulin stimulation in vitro. Because insulin promotes lipogenesis in hepatocytes, we hypothesized that the variant was causally linked to FLD in the family. To test this hypothesis, we used CRISPR/Cas9 technology to replace the corresponding tyrosine in mouse INSR with histidine (Y1345H). No significant differences were found in hepatic insulin signaling, as assessed by phosphorylation of INSR or AKT levels or in activation of the insulin-responsive transcription factor SREBP-1c. Glucose tolerance and hepatic triglyceride (TG) content in Insr1345H/H mice fed a chow diet or diets rich in fat, sucrose or fructose did not differ significantly from WT littermates. Thus, our studies in mice failed to support the notion that INSR (Y1355H) is causally related to FLD in the family or that phosphorylation of this residue alters hepatic TG metabolism.

Functional analysis of an R311C variant of Ca2+ -calmodulin-dependent protein kinase kinase-2 (CaMKK2) found as a de novo mutation in a patient with bipolar disorder.

Loss-of-function mutations in the gene encoding the calcium-calmodulin (Ca2+ -CaM)-dependent protein kinase kinase-2 (CaMKK2) enzyme are linked to bipolar disorder. Recently, a de novo arginine to cysteine (R311C) mutation in CaMKK2 was identified from a whole exome sequencing study of bipolar patients and their unaffected parents. The aim of the present study was to determine the functional consequences of the R311C mutation on CaMKK2 activity and regulation by Ca2+ -CaM. The effects of the R311C mutation on CaMKK2 activity and Ca2+ -CaM activation were examined using a radiolabeled adenosine triphosphate (ATP) kinase assay. We performed immunoblot analysis to determine whether the R311C mutation impacts threonine-85 (T85) autophosphorylation, an activating phosphorylation site on CaMKK2 that has also been implicated in bipolar disorder. We also expressed the R311C mutant in CaMKK2 knockout HAP1 cells and used immunoblot analysis and an MTS reduction assay to study its effects on Ca2+ -dependent downstream signaling and cell viability, respectively. The R311C mutation maps to the conserved HRD motif within the catalytic loop of CaMKK2 and caused a marked reduction in kinase activity and Ca2+ -CaM activation. The R311C mutation virtually abolished T85 autophosphorylation in response to Ca2+ -CaM and exerted a dominant-negative effect in cells as it impaired the ability of wild-type CaMKK2 to initiate downstream signaling and maintain cell viability. The highly disruptive, loss-of-function impact of the de novo R311C mutation in human CaMKK2 provides a compelling functional rationale for being considered a potential rare monogenic cause of bipolar disorder.

The catalytic subunit of DNA-PK has a unique function in inflammation independently of Ku70 and DNA repair: a new opportunity to target the enzyme without interfering with DNA repair

Abstract Our laboratory demonstrated a critical role for DNA-dependent protein kinase (DNA-PK) in asthma pathogenesis via modulating the pertinent immune responses. DNA-PK is DNA repair enzyme composed of a catalytic subunit (DNA-PKcs) and two DNA-binding subunits (Ku70 and Ku80). Human cells express high levels of DNA-PK, surprisingly such high levels do not confer increased ability to repair DNA damage. Here we show that the role of DNA-PK in promoting inflammatory responses is independent of its function in the DNA repair. We examined the effect(s) of partial depletion of Ku70 on Ovalbumin (OVA) induced lung inflammation in mouse model of the disease. Of note, depletion of Ku70 by gene heterozygosity causes deficiency in DNA-PK-dependent DNA repair. Unlike the protective effects provided by DNA-PKcs gene heterozygosity, Ku70 heterozygosity did not alter the OVA-induced eosinophilia, mucus hypersecretion, Th2 cytokines production, or OVA-specific IgE upon OVA challenge. Interestingly, Ku70 heterozygosity enhanced methacholine-induced AHR over that of WT mice. Using a cell culture system, we demonstrate that while IL-4 and TNF-α are potent inducers of DNA-PKcs, such activation did not coincide with any detectable DNA damage or repair responses. Remarkably, while Ku70−/− blocked DNA-PKcs autophosphorylation in response to the DNA damage agent, etoposide, it did not affect the kinase in response to TNFα. Our findings suggest that the mechanism by which DNA-PK functions in inflammation is completely unrelated to its role in DNA repair, thus, unraveling a completely novel function for the kinase. More importantly, this provides a window of opportunity to target DNA-PK in inflammatory diseases without interfering with DNA repair processes.

DR-03 * MECHANISMS THAT CAUSE RESISTANCE TO EGFR TARGETED THERAPIES IN GLIOMA

The epidermal growth factor receptor (EGFR), or its expression, is altered in 57% of glioma by mutation, rearrangement, altered splicing and/or focal amplification, suggesting that EGFR should be a prime target for therapy. However, attempts to therapeutically target EGFR in patients with glioma have so far failed. The fundamental reasons why EGFR inhibitors are so ineffective against glioma remain largely unknown. We used three approaches to address this question. First, we derived a range of early passage neurosphere cell lines from glioma patients. These cell lines express different forms of the mutated EGFR, including the most common alterations in glioma: the autoactivating deletion mutant EGFRvIII and the extracellular domain (ECD) point mutation A289V. Through exonic sequencing and signaling pathway analysis of these cell lines, several of which are resistant to EGFR-targeted therapeutics, we showed that activation of the ras/MEK/ERK signaling pathway led to innate resistance to EGFR-targeted drugs (to both tyrosine kinase inhibitors and EGFR-specific antibodies). Likewise, engagement of neurosphere cell lines with extracellular matrix proteins such as laminin caused resistance to EGFR-targeted therapeutics. Second, we analyzed a range of EGFR ECD cysteine mutants identified in glioma patient samples. All four mutants analyzed were found on the cell surface as inactive, preformed dimers. However, all receptor mutants had enhanced activation, as measured by autophosphorylation in response to EGFR ligands. Importantly, expression of these mutants in U87MG glioma xenografts rendered the tumors more susceptible to EGFR-targeted therapeutics than xenografts expressing wild-type EGFR. Last, we show that EGFRvIII partly mediates its tumorigenicity through promiscuous activation of other receptor tyrosine kinases, such as MET, through a FAK scaffold. Co-targeting the kinase activity of FAK significantly increased the antitumor activity of EGFR-targeted therapeutics. These studies provide new insight into the mechanisms of resistance and sensitivity to EGFR-based drugs in glioma.

Glycosylation at Asn211 Regulates the Activation State of the Discoidin Domain Receptor 1 (DDR1)

Discoidin domain receptor 1 (DDR1) belongs to a unique family of receptor tyrosine kinases that signal in response to collagens. DDR1 undergoes autophosphorylation in response to collagen binding with a slow and sustained kinetics that is unique among members of the receptor tyrosine kinase family. DDR1 dimerization precedes receptor activation suggesting a structural inhibitory mechanism to prevent unwarranted phosphorylation. However, the mechanism(s) that maintains the autoinhibitory state of the DDR1 dimers is unknown. Here, we report that N-glycosylation at the Asn(211) residue plays a unique role in the control of DDR1 dimerization and autophosphorylation. Using site-directed mutagenesis, we found that mutations that disrupt the conserved (211)NDS N-glycosylation motif, but not other N-glycosylation sites (Asn(260), Asn(371), and Asn(394)), result in collagen I-independent constitutive phosphorylation. Mass spectrometry revealed that the N211Q mutant undergoes phosphorylation at Tyr(484), Tyr(520), Tyr(792), and Tyr(797). The N211Q traffics to the cell surface, and its ectodomain displays collagen I binding with an affinity similar to that of the wild-type DDR1 ectodomain. However, unlike the wild-type receptor, the N211Q mutant exhibits enhanced receptor dimerization and sustained activation upon ligand withdrawal. Taken together, these data suggest that N-glycosylation at the highly conserved (211)NDS motif evolved to act as a negative repressor of DDR1 phosphorylation in the absence of ligand. The presence of glycan moieties at that site may help to lock the collagen-binding domain in the inactive state and prevent unwarranted signaling by receptor dimers. These studies provide a novel insight into the structural mechanisms that regulate DDR activation.

Construction of AgrC Proteoliposomes by Detergent-Mediated Method

To respond appropriately to different environmental changes, bacteria have evolved two-component signal transduction systems (tcsts), which are absent in mammals (including human beings). a typical tcst consisted of a sensor kinase (histidine kinase, hk) and a response regulator (rr). hk is capable of autophosphorylation in response to an environmental signal, while rr interacts with the phosphorylated hk. membrane protein agrc is a sensor kinase of a tcst from staphylococcus aureus. illumination of signal transduction mechanism is of great significance to solve the problem of bacterial resistance. at present, the main bottleneck of membrane protein is the difficulties in obtaining large quantities of sufficiently pure and functional protein. the target protein was overexpressed in escherichia coli, solubilized from cell membranes and purified in detergent micelles. this series of steps tend to lead to destabilizations of membrane protein and loss of function. in this study, agrc was incorporated into liposomes by a detergent-mediated method. for standard incorporation, protein solution was added to detergent-lipid suspension containing lipid at 2.5 mmol/l at a lipid-to-protein ratio of 300 (mol/mol). for slow detergent removal, successive additions of small amounts of beads at bio-bead-to-detergent ratios of 2 (w/w) will allow the removal of the detergent, resulting in formation of proteoliposome. the structure, morphology and average diameter of liposomes and proteoliposomes were characterized by transmission electron microscopy (tem) and dynamic light scattering (dls), respectively. sucrose density gradient centrifugation was employed to separate proteoliposomes. the result showed efficiency of protein incorporation and liposomes recovery reached 80% and 90%, respectively. thiol reagent labeling test showed the cytoplasmic domain of agrc was almost exclusively oriented towards the inside of the liposome vesicles. in vitro phosphorylation experiments showed that kinase activity of agrc in proteoliposomes was significantly higher than in detergent micelles. proteoliposomes could be stored for two weeks with little loss of function. preparation of proteoliposome not only solves the instability problem of membrane proteins, but also provides a new approach of the study of membrane protein structure, function and signal transduction mechanism in vitro.

The Novel ATP-Competitive Inhibitor of the MET Hepatocyte Growth Factor Receptor EMD1214063 Displays Inhibitory Activity against Selected MET-Mutated Variants

The receptor tyrosine kinase MET is a prime target in clinical oncology due to its aberrant activation and involvement in the pathogenesis of a broad spectrum of malignancies. Similar to other targeted kinases, primary and secondary mutations seem to represent an important resistance mechanism to MET inhibitors. Here, we report the biologic activity of a novel MET inhibitor, EMD1214063, on cells that ectopically express the mutated MET variants M1268T, Y1248H, H1112Y, L1213V, H1112L, V1110I, V1206L, and V1238I. Our results show a dose-dependent decrease in MET autophosphorylation in response to EMD1214063 in five of the eight cell lines (IC50 2-43 nmol/L). Blockade of MET by EMD1214063 was accompanied by a reduced activation of downstream effectors in cells expressing EMD1214063-sensitive mutants. In all sensitive mutant-expressing lines, EMD1214063 altered cell-cycle distribution, primarily with an increase in G1 phase. EMD1214063 strongly influenced MET-driven biologic functions, such as cellular morphology, MET-dependent cell motility, and anchorage-independent growth. To assess the in vivo efficacy of EMD1214063, we used a xenograft tumor model in immunocompromised mice bearing NIH3T3 cells expressing sensitive and resistant MET-mutated variants. Animals were randomized for the treatment with EMD1214063 (50 mg/kg/d) or vehicle only. Remarkably, five days of EMD1214063 treatment resulted in a complete regression of the sensitive H1112L-derived tumors, whereas tumor growth remained unaffected in mice with L1213V tumors and in vehicle-treated animals. Collectively, the current data identifies EMD1214063 as a potent MET small-molecule inhibitor with selective activity towards mutated MET variants.

Abstract 3329: The DNA-PK inhibitor, NU7441, sensitises cancer cells to microtubule-targeting agents and modulates vincristine efflux in multidrug resistant cells.

Abstract Microtubules are involved in the formation of the mitotic spindle, and correct separation of the sister chromatids at mitosis is essential for maintaining genomic stability. Microtubule-targeting drugs arrest rapidly-dividing cells and are important chemotherapeutic agents, but the development of multidrug-resistance (MDR) is a serious clinical problem. Along with its role in DNA double-strand break repair, DNA-dependent protein kinase (DNA-PK) has also been shown to play an important role in stabilising spindle formation and preventing mitotic catastrophe (Shang et al, Cancer Res. 2010). In light of this novel finding, we hypothesised that inhibition of DNA-PK would sensitise cells to microtubule-targeting agents. Pilot studies showed that inhibition of DNA-PK by the selective inhibitor, NU7441, caused greater sensitisation to vincristine in multidrug-resistant cells compared to parental cells. Here, we further investigate the role of DNA-PK in response to microtubule-targeting drugs using a novel series of NU7441 analogues, and investigate whether these inhibitors interact with the efflux transporter, P-glycoprotein. In growth inhibition studies, CCRF-CEM leukaemia cells were sensitised 1.4-fold to vincristine by NU7441 (1μM) whereas their vincristine-resistant derivative CCRF-CEM VCR/R cells were sensitised 8-fold to vincristine. DNA-PK-deficient M059J glioblastoma cells were 2-fold more sensitive to docetaxel than DNA-PK-proficient cells, and NU7441 sensitised only DNA-PK-proficient M059J-Fus1 cells to docetaxel. CCRF-CEM VCR/R cells have a higher basal level of autophosphorylation of DNA-PKcs (Western blotting) than the CCRF-CEM cells and autophosphorylation in response to vincristine was abrogated by NU7441. To investigate the effects of NU7441 on drug transport, a doxorubicin fluorescence assay showed that in P-glycoprotein-overexpressing canine kidney MDCKII-MDR1 cells, 1μM NU7441 increased doxorubicin cellular fluorescence 16-fold by blocking drug efflux via P-glycoprotein. NU7441 and 3 structurally-related compounds (NU7742 (inactive NU7441 analogue), DRN1 (DNA-PK-inhibitory atropisomeric NU7441 derivative) or DRN2 (DNA-PK non-inhibitory atropisomeric NU7441 derivative)) all increased intracellular vincristine accumulation in the CCRF-CEM VCR/R cells to a level similar to verapamil, as measured by LC-MS. However, the non-inhibitory compound NU7742 was 5-fold less effective at sensitising CCRF-CEM VCR/R cells to vincristine than NU7441 (p=0.008). The novel finding that DNA-PK inhibition sensitises cells to microtubule-targeting agents suggests that along with its function in DNA repair, this enzyme plays a role in the mitotic spindle checkpoint. Additionally, in multidrug-resistant cells, NU7441 is able to block drug efflux via interaction with P-glycoprotein. Citation Format: Emily Mould, Philip Berry, David Jamieson, Christopher Hill, Niu Tan, Sarah Elliott, Barbara Durkacz, David Newell, Elaine Willmore. The DNA-PK inhibitor, NU7441, sensitises cancer cells to microtubule-targeting agents and modulates vincristine efflux in multidrug resistant cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3329. doi:10.1158/1538-7445.AM2013-3329

CENP-E–dependent BubR1 autophosphorylation enhances chromosome alignment and the mitotic checkpoint

How the state of spindle microtubule capture at the kinetochore is translated into mitotic checkpoint signaling remains largely unknown. In this paper, we demonstrate that the kinetochore-associated mitotic kinase BubR1 phosphorylates itself in human cells and that this autophosphorylation is dependent on its binding partner, the kinetochore motor CENP-E. This CENP-E-dependent BubR1 autophosphorylation at unattached kinetochores is important for a full-strength mitotic checkpoint to prevent single chromosome loss. Replacing endogenous BubR1 with a nonphosphorylatable BubR1 mutant, as well as depletion of CENP-E, the BubR1 kinase activator, results in metaphase chromosome misalignment and a decrease of Aurora B-mediated Ndc80 phosphorylation at kinetochores. Furthermore, expressing a phosphomimetic BubR1 mutant substantially reduces the incidence of polar chromosomes in CENP-E-depleted cells. Thus, the state of CENP-E-dependent BubR1 autophosphorylation in response to spindle microtubule capture by CENP-E is important for kinetochore function in achieving accurate chromosome segregation.

Regulation of Discoidin Domain Receptor‐1 by Membrane‐type 1 Matrix Metalloproteinase (MT1‐MMP)

Objective We investigated the effects of membrane type-matrix metalloproteinases (MT-MMPs) on the activation of discoidin domain receptors (DDRs), which are receptor tyrosine kinases (RTKs) that undergo receptor autophosphorylation in response to collagen. Methods Proteases were co-expressed with DDR1a or DDR1b and receptor phosphorylation in response to collagen was examined. Results we found that the transmembrane MT-MMPs, MT1-(MMP14), MT2- (MMP15), and MT3- (MMP16) MMP but not other members of the MT-MMP subfamily hinder collagen-dependent DDR1a/b activation and promote cleavage of the receptor ectodomain. Two soluble collagenases, MMP1 and MMP13, and a chimeric MT1-MMP containing the catalytic domain of MMP1 had no effect on DDR1 activation or cleavage, suggesting a specific effect of MT-MMPs on DDR1 regulation. T47D breast cancer cells expressing recombinant MT1-MMP exhibit reduced collagen-dependent phosphorylation and increased cleavage of endogenous DDR1. Conclusion These results show that DDR1a/b activation is specifically regulated by MT-MMPs and suggests that MT-MMP/DDR interactions may play a role in regulation of cell-collagen interactions during cell migration and invasion.

Regulation of PKA catalytic activity by phosphorylation at multiple sites during the fermentative‐respiratory transition in Saccharomyces cerevisiae

In the AGC family of serine/threonine kinases, the activities of catalytic subunits are regulated by multiple phosphorylation both in the activation loop and at allosteric sites. In yeast, PKA has three catalytic subunits, TPK1, TPK2 and TPK3. We describe a transient change in the phosphorylation state of Tpk1p during in vivo cAMP pathway activation evoked by glucose addition to glycerol-growing cells. Phosphorylation of Tpk1 increases its Ksp toward kemptide. Genetic and in vitro kinetic experiments indicate an intramolecular phosphorylation mechanism for Tpk1 during glucose stimulus. PDK1 controls several protein kinases through phosphorylation on a conserved Thr in the activation loop. S.cerevisiae contains three PDK1 homologues: Pkh1, Pkh2 and Pkh3. We show that Pkh1 is capable of phosphorylating Thr241 of Tpk1 in vivo permitting the interaction with the regulatory subunit Bcy1. Using pkhD398G, pkh2 delta mutant we studied the effect of inactivation of Pkh activity over several phenotypes controlled by Tpk1. These experiments point to the Pkh kinase as an upstream negative regulator of the cAMP-PKA pathway in yeast. The results suggest at least two phosphorylation pathways operating on TPK1: i) Tpk1 itself through autophosphorylation in response to glucose stimulus and ii) Tpk1 through Thr241 phosphorylation by Pkh. Research supported by University of Buenos Aires, CONICET and ANPCYT grants.

Myoferlin Regulates Vascular Endothelial Growth Factor Receptor-2 Stability and Function

Myoferlin and dysferlin are members of the ferlin family of membrane proteins. Recent studies have shown that mutation or genetic disruption of myoferlin or dysferlin promotes muscular dystrophy-related phenotypes in mice, which are the result of impaired plasma membrane integrity. However, no biological functions have been ascribed to myoferlin in non-muscle tissues. Herein, using a proteomic analysis of endothelial cell (EC) caveolae/lipid raft microdomains we identified myoferlin in these domains and show that myoferlin is highly expressed in ECs and vascular tissues. The loss of myoferlin results in lack of proliferation, migration, and nitric oxide (NO) release in response to vascular endothelial growth factor (VEGF). Western blotting and surface biotinylation experiments show that loss of myoferlin reduces the expression level and autophosphorylation of VEGF receptor-2 (VEGFR-2) in native ECs. In a reconstituted cell system, transfection of myoferlin increases VEGFR-2 membrane expression and autophosphorylation in response to VEGF. In vivo, VEGFR-2 levels and VEGF-induced permeability are impaired in myoferlin-deficient mice. Mechanistically, myoferlin forms a complex with dynamin-2 and VEGFR-2, which prevents CBL-dependent VEGFR-2 polyubiquitination and proteasomal degradation. These data are the first to report novel biological activities for myoferlin and reveal the role of membrane integrity to VEGF signaling.

In-frame deletion in the EGF receptor alters kinase inhibition by gefitinib

The existence of an in-frame deletion mutant correlates with the sensitivity of lung cancers to EGFR (epidermal growth factor receptor)-targeted tyrosine kinase inhibitors. We reported previously that the in-frame 15-bp deletional mutation (delE746-A750 type deletion) was constitutively active in cells. Kinetic parameters are important for characterizing an enzyme; however, it remains unclear whether the kinetic parameters of deletion mutant EGFR are similar to those of wild-type EGFR. We analysed autophosphorylation in response to ATP and inhibition of gefitinib for deletion mutant EGFR and wild-type EGFR. Kinetic studies, examining autophosphorylation, were carried out using EGFR fractions extracted from 293-pDelta15 and 293-pEGFR cells transfected with deletion mutant EGFR and wild-type EGFR respectively. We demonstrated the difference in activities between unstimulated wild-type (K(m) for ATP=4.0+/-0.3 microM) and mutant EGFR (K(m) for ATP=2.5+/-0.2 microM). There was no difference in K(m) values between EGF-stimulated wild-type EGFR (K(m) for ATP=1.9+/-0.1 microM) and deletion mutant EGFR (K(m) for ATP=2.2+/-0.2 microM). These results suggest that mutant EGFR is active without ligand stimulation. The K(i) value for gefitinib of the deletion mutant EGFR was much lower than that of wild-type EGFR. These results suggest that the deletion mutant EGFR has a higher affinity for gefitinib than wild-type EGFR.

Cytometric assessment of DNA damage in relation to cell cycle phase and apoptosis

Reviewed are the methods aimed to detect DNA damage in individual cells, estimate its extent and relate it to cell cycle phase and induction of apoptosis. They include the assays that reveal DNA fragmentation during apoptosis, as well as DNA damage induced by genotoxic agents. DNA fragmentation that occurs in the course of apoptosis is detected by selective extraction of degraded DNA. DNA in chromatin of apoptotic cells shows also increased propensity to undergo denaturation. The most common assay of DNA fragmentation relies on labelling DNA strand breaks with fluorochrome-tagged deoxynucleotides. The induction of double-strand DNA breaks (DSBs) by genotoxic agents provides a signal for histone H2AX phosphorylation on Ser139; the phosphorylated H2AX is named gammaH2AX. Also, ATM-kinase is activated through its autophosphorylation on Ser1981. Immunocytochemical detection of gammaH2AX and/or ATM-Ser1981(P) are sensitive probes to reveal induction of DSBs. When used concurrently with analysis of cellular DNA content and caspase-3 activation, they allow one to correlate the extent of DNA damage with the cell cycle phase and with activation of the apoptotic pathway. The presented data reveal cell cycle phase-specific patterns of H2AX phosphorylation and ATM autophosphorylation in response to induction of DSBs by ionizing radiation, topoisomerase I and II inhibitors and carcinogens. Detection of DNA damage in tumour cells during radio- or chemotherapy may provide an early marker predictive of response to treatment.

Selective modulation of type 1 insulin-like growth factor receptor signaling and functions by beta1 integrins.

We show here that β1 integrins selectively modulate insulin-like growth factor type I receptor (IGF-IR) signaling in response to IGF stimulation. The β1A integrin forms a complex with the IGF-IR and insulin receptor substrate-1 (IRS-1); this complex does not promote IGF-I mediated cell adhesion to laminin (LN), although it does support IGF-mediated cell proliferation. In contrast, β1C, an integrin cytoplasmic variant, increases cell adhesion to LN in response to IGF-I and its down-regulation by a ribozyme prevents IGF-mediated adhesion to LN. Moreover, β1C completely prevents IGF-mediated cell proliferation and tumor growth by inhibiting IGF-IR auto-phosphorylation in response to IGF-I stimulation. Evidence is provided that the β1 cytodomain plays an important role in mediating β1 integrin association with either IRS-1 or Grb2-associated binder1 (Gab1)/SH2-containing protein-tyrosine phosphate 2 (Shp2), downstream effectors of IGF-IR: specifically, β1A associates with IRS-1 and β1C with Gab1/Shp2. This study unravels a novel mechanism mediated by the integrin cytoplasmic domain that differentially regulates cell adhesion to LN and cell proliferation in response to IGF.

Phosphorylation of SMC1 is a critical downstream event in the ATM-NBS1-BRCA1 pathway.

The ATM protein kinase is activated by intermolecular autophosphorylation in response to DNA damage and initiates cellular signaling pathways that facilitate cell survival and reduce chromosomal breakage. Here, we show that NBS1 and BRCA1 are required for the recruitment of previously activated ATM to the sites of DNA breaks after ionizing irradiation, and that this recruitment is required for the phosphorylation of SMC1 by ATM. To explore the functional importance of SMC1 phosphorylation, murine cells were generated, in which the two damage-induced phosphorylation sites in SMC1 are mutated. Although these cells demonstrate normal phosphorylation and focus formation of ATM, NBS1, and BRCA1 proteins after IR, they exhibit a defective S-phase checkpoint, decreased survival, and increased chromosomal aberrations after DNA damage. These observations suggest that many of the abnormal stress responses seen in cells lacking ATM, NBS1, or BRCA1 result from a failure of ATM migration to sites of DNA breaks and a resultant lack of SMC1 phosphorylation.

Cellular and Subcellular Localization of Phototropin 1

Phototropin 1 (phot1) is a Ser/Thr photoreceptor kinase that binds two molecules of flavin mononucleotide as its chromophores and undergoes autophosphorylation in response to blue light. Phot1 is plasma membrane associated and, as with phot2, has been shown to function as a photoreceptor for phototropism, blue light-induced chloroplast movement, and blue light-induced stomatal opening. Phot1 likely also plays a redundant role with phot2 in regulating the rate of leaf expansion. Understanding the mechanism(s) by which phot1 initiates these four different responses requires, at minimum, knowledge of where the photoreceptor is located. Therefore, we transformed a phot1 null mutant of Arabidopsis with a construct encoding translationally fused phot1-green fluorescent protein (GFP) under the control of the endogenous PHOT1 promoter and investigated its cellular and subcellular distribution. This PHOT1-GFP construct complements the mutant phenotype, restoring second positive curvature. Phot1 is expressed strongly in dividing and elongating cortical cells in the apical hook and in the root elongation zone in etiolated seedlings. It is localized evenly to the plasma membrane region in epidermal cells but is confined largely to the plasma membrane region of the transverse cell walls in the cortical cells of both root and hypocotyl. It is found at both apical and basal ends of these cortical cells. In light-grown plants, phot1-GFP is localized largely in the plasma membrane regions adjacent to apical and basal cell end walls in the elongating inflorescence stem, where the photoreceptor is expressed strongly in the vascular parenchyma and leaf vein parenchyma. Phot1 also is localized to the plasma membrane region of leaf epidermal cells, mesophyll cells, and guard cells, where its distribution is uniform. Although phot1 is localized consistently to the plasma membrane region in etiolated seedlings, a fraction becomes released to the cytoplasm in response to blue light. Possible relationships between observed phot1 distribution and the various physiological responses activated by blue light are discussed.

GFP-FRNK disrupts focal adhesions and induces anoikis in neonatal rat ventricular myocytes.

Focal adhesion kinase (FAK) is a nonreceptor protein tyrosine kinase involved in adhesion-dependent signal transduction. FAK is highly expressed in cultured neonatal rat ventricular myocytes (NRVMs) and undergoes tyrosine autophosphorylation in response to cell adhesion, stretch, and growth factor stimulation. We previously showed that inhibition of FAK phosphorylation by adenovirally mediated overexpression of FRNK (the autonomously expressed C-terminal domain of FAK) prevented endothelin-1 (ET)-induced NRVM hypertrophy. One question raised by these studies was whether FRNK localized to focal adhesions and displaced FAK from sites required for downstream signaling. Therefore, we constructed a replication-defective adenovirus encoding a GFP-FRNK fusion protein (Adv-GFP-FRNK) and examined its effects on NRVM cytoarchitecture and signaling. Uninfected NRVMs contained small amounts of endogenous FRNK. NRVMs infected with Adv-GFP-FRNK expressed much larger amounts of a 66-/68-kDa protein that localized to costameres and focal adhesions. GFP-FRNK overexpression suppressed basal and ET-induced FAK phosphorylation and also inhibited ET-induced phosphorylation of PYK2, the other member of the FAK family of nonreceptor protein tyrosine kinases. In contrast, GFP-FRNK overexpression did not prevent ET-induced ERK, JNK, or p70S6K phosphorylation. Furthermore, GFP-FRNK resulted in the loss of detectable FAK and paxillin in focal adhesions, which was accompanied by reduced levels of total paxillin and, ultimately, cell detachment and apoptosis. We conclude that FRNK functions as a dominant-negative inhibitor of adhesion-dependent signaling by displacing FAK from focal adhesions and interfering with the anchorage of NRVMs that is necessary for cell survival, a process known as anoikis.

Phosphorylation of Threonine 68 Promotes Oligomerization and Autophosphorylation of the Chk2 Protein Kinase via the Forkhead-associated Domain

Phosphorylation of Thr-68 by the ataxia telangiectasia-mutated is necessary for efficient activation of Chk2 when cells are exposed to ionizing radiation. By an unknown mechanism, this initial event promotes additional autophosphorylation events including modifications of Thr-383 and Thr-387, two amino acid residues located within the activation loop segment within the Chk2 catalytic domain. Chk2 and related kinases possess one or more Forkhead-associated (FHA) domains that are phosphopeptide-binding modules believed to be crucial for their checkpoint control activities. We show that the Chk2 FHA domain is dispensable for Thr-68 phosphorylation but necessary for efficient autophosphorylation in response to ionizing radiation. Phosphorylation of Thr-68 promotes oligomerization of Chk2 by serving as a specific ligand for the FHA domain of another Chk2 molecule. In addition, Chk2 phosphorylates its own FHA domain, and this modification reduces its affinity for Thr-68-phosphorylated Chk2. Thus, activation of Chk2 in irradiated cells may occur through oligomerization of Chk2 via binding of the Thr-68-phosphorylated region of one Chk2 to the FHA domain of another. Oligomerization of Chk2 may therefore increase the efficiency of trans-autophosphorylation resulting in the release of active Chk2 monomers that proceed to enforce checkpoint control in irradiated cells.

Mechanisms of decidualization

AbstractThe uterus is composed of heterogeneous cell types that undergo cyclic synchronized waves of proliferation and differentiation in response to the rise and fall of ovarian oestrogen and progesterone. The spatial and temporal diversity in cellular responses to ovarian hormones within a given endometrial cell compartment is thought to be effected by locally released factors. These endometrial polypeptides bind to specific cell surface receptors on target cells, resulting in activation of signal transduction pathways by way of coupling to GTP-binding proteins (G proteins), or through autophosphorylation in response to conformational changes induced by the binding of ligand. Within this paradigm, the highly complex and coordinated expression of decidua-specific genes by differentiating endometrial stroma cells during the late secretory phase of the menstrual cycle could be regarded as the result of the convergence of liganded steroid hormone receptors and specific activated cytoplasmatic signalling pathways.