Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: UMLS:C0004135 (ATM)
 13,001 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The glucose transporter isoform, GLUT4, has been expressed in Chinese hamster clones and its subcellular trafficking has been determined following labelling at the cell surface with the impermeant bis-mannose photolabel, 2-N-(1-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis(D-mannos -4-yloxy)-2-propylamine (ATM-BMPA). ATM-BMPA-tagged GLUT4 leaves the cell surface rapidly and equilibrates to give an internal/surface distribution ratio of approx. 3.5 after 60 min. GLUT4 in which the N-terminal phenylalanine-5 and glutamine-6 are mutated to alanine-N-(FQ-AA) and in which the C-terminal leucine-489 and -490 are mutated to alanine C-(LL-AA) have low internal/surface ratios of 0.64 and 1.24 respectively. If all cell-surface transporters are able to recycle, as would be the case for a two-pool recycling model with a single intracellular pool, then analysis suggests that the wild-type GLUT4 distribution ratio is dependent on endocytosis and exocytosis rate constants of 0.074 and 0.023 min(-1). These values are similar, but not identical, to those found for GLUT4 trafficking in adipocytes. The distribution of the N-(FQ-AA) transporter appears to be due to a decrease in endocytosis with reduced intracellular retention, while the distribution of the C-(LL_AA) transporter appears to be mainly due to poor intracellular retention. These results are also considered in terms of a consecutive intracellular pool model in which GLUT4 targeting domains alter the distribution between recycling endosomes and a slowly recycling compartment. In this case the more rapid apparent exocytosis of the mutated GLUT4 is due to their failure to reach a slowly recycling compartment with a consequent return to the plasma membrane by default. It is suggested that overexpression of transporters increases the proportion that are recycled in this way. Wortmannin is shown to decrease glucose transport activity and cell-surface photolabelled transporters in a manner consistent with an inhibition of transporter recycling. Studies on the rate of loss of transport activity and ATB-BMPA-tagged transporter in wortmannin-treated cells confirm that the N-(FQ-AA) mutant is endocytosed more slowly than the wild-type GLUT4. Taken together, these results suggest that the mutation at either the N- or the C-terminal domain can reduce movement to a slowly recycling intracellular compartment but that neither domain alone is entirely sufficient to produce wild-type GLUT4 trafficking behaviour.
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PMID:Subcellular trafficking kinetics of GLU4 mutated at the N- and C-terminal. 867 Jan 1

A fifth transmembrane domain lysine residue is conserved in both the type 1 (AT1) and type 2 (AT2) angiotensin II (AngII) receptors. This lysine (Lys199) is believed to play a critical role in peptide binding for the AT1 receptor. To evaluate its possible role in the AT2 receptor, the analogous AT2 residue (Lys199) was changed to glutamine. This mutation greatly reduced the affinity for both 125I-AngII and 125I-Sar1,Ile8-AngII and abolished binding to the non-peptide 125I-PD122979. These data indicate that despite a relatively low homology of 34%, some commonalities in the binding mechanism for AngII may exist between the two subtypes.
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PMID:Mutation of a conserved fifth transmembrane domain lysine residue (Lys215) attenuates ligand binding in the angiotensin II type 2 receptor. 942 26

Studies on ligand-receptor interaction of Angiotensin II (Ang II) receptor type 1 have shown that for peptidic ligands to bind this receptor they must interact via their C-terminal carboxylate group to the positively charged side chain of the Lysine residue 199 located in the fifth transmembrane domain of this receptor. In the Ang II receptor type AT2, this Lysine residue is conserved at position 215 in the fifth transmembrane domain. To determine the specific mechanism of ligand binding to the Angiotensin II receptor type AT2, mutated AT2 receptors were generated in which the Lys215 was replaced with glutamic acid, glutamine, alanine and arginine. The ability of these mutated receptors to bind peptidic ligands 125I-[Sar1-Ile8]Ang II (non-specific for AT2 receptor type), 125I-CGP42112A (AT2 receptor specific) and the non-peptidic ligand PD123319 (AT2 receptor specific) was evaluated by expressing these receptors in Xenopus oocytes and performing binding assays. The Lys215Glu and Lys215Gln mutants of AT2 receptor lost their affinity to 125I-[Sar1-Ile8]Ang II, but retained their affinity to 125I-CGP42112A and PD123319. In contrast, Lys215Arg mutant retained its affinity to 125I-[Sar1-Ile8]Ang II, but exhibited lower affinity to 125I-CGP42112A. The Lys215Ala mutant lost its affinity to both 125I-[Sar1-Ile8]Ang II and 125I-CGP42112A. These results suggest that the binding mechanism of 125I-[Sar1-Ile8]Ang II to AT2 receptor is similar to that of AT1 receptor since an amino acid with positively charged side chain (Lys or Arg) located in the fifth transmembrane domain is required for this ligand to bind AT2 receptor. In contrast, although CGP42112A is a peptidic ligand, it does not require an interaction between its C-terminal carboxylate group and the positively charged side-chain of an amino acid in the fifth transmembrane domain for its binding to AT2 receptor.
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PMID:Role of Lys215 located in the fifth transmembrane domain of the AT2 receptor in ligand-receptor interaction. 953 73

Angiotensin II receptor subtypes AT1 and AT2 are proteins with seven transmembrane domain (TMD) topology and share 34% homology. It was shown that His256, located in the sixth TMD of the AT1 receptor, is needed for the agonist activation by the Phe8 side chain of angiotensin II, although replacing this residue with arginine or glutamine did not significantly alter the affinity binding of the receptor. We hypothesized that the His273 located in the sixth transmembrane domain of the AT2 receptor may play a similar role in the functions of the AT2 receptor, although this residue was not identified as a conserved residue in the initial homology comparisions. Therefore, we replaced His273 of the AT2 receptor with arginine or glutamine and analyzed the ligand-binding properties of the mutant receptors using Xenopus oocytes as an expression system. Our results suggested that the AT2 receptor mutants His273Arg and His273 Glu have lost their affinity to [125I-Sar1-Ile8]Ang II, a peptidic ligand that binds both the AT1 and AT2 receptors and to 125I-CGP42112A, a peptidic ligand that binds specifically to the AT2 receptor. Thus, His273 located in the sixth TMD of the AT2 receptor seems to play an important role in determining the binding properties of this receptor. Moreover, these results along with our previous observation that the Lys215 located in the 5th TMD of the AT2 receptor is essential for its high affinity binding to [125I-Sar1-Ile8]Ang II indicate that key amino acids located in the 5th and 6th TMDs of the AT2 receptor are needed for high affinity binding of the AT2 to its ligands.
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PMID:Role of the His273 located in the sixth transmembrane domain of the angiotensin II receptor subtype AT2 in ligand-receptor interaction. 1020 47

The Brca1 (breast cancer gene 1) tumor suppressor protein is phosphorylated in response to DNA damage. Results from this study indicate that the checkpoint protein kinase ATM (mutated in ataxia telangiectasia) was required for phosphorylation of Brca1 in response to ionizing radiation. ATM resides in a complex with Brca1 and phosphorylated Brca1 in vivo and in vitro in a region that contains clusters of serine-glutamine residues. Phosphorylation of this domain appears to be functionally important because a mutated Brca1 protein lacking two phosphorylation sites failed to rescue the radiation hypersensitivity of a Brca1-deficient cell line. Thus, phosphorylation of Brca1 by the checkpoint kinase ATM may be critical for proper responses to DNA double-strand breaks and may provide a molecular explanation for the role of ATM in breast cancer.
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PMID:Requirement of ATM-dependent phosphorylation of brca1 in the DNA damage response to double-strand breaks. 1061 May 23

Fission yeast Cds1 is phosphorylated and activated when DNA replication is interrupted by nucleotide starvation or DNA damage. Cds1 enforces the S-M checkpoint that couples mitosis (M) to the completion of DNA synthesis (S). Cds1 also controls replicational stress tolerance mechanisms. Cds1 is regulated by a group of proteins that includes Rad3, a kinase related to human checkpoint kinase ATM (ataxia telangiectasia mutated). ATM phosphorylates serine or threonine followed by glutamine (SQ or TQ). Here we show that in vitro, Rad3 and ATM phosphorylate the N-terminal domain of Cds1 at the motif T(11)Q(12). Substitution of threonine-11 with alanine (T11A) abolished Cds1 activation that occurs when DNA replication is inhibited by hydroxyurea (HU) treatment. The cds1-T11A mutant was profoundly sensitive to HU, although not quite as sensitive as a cds1(-) null mutant. Cds1(T11A) was unable to enforce the S-M checkpoint. These results strongly suggest that Rad3-dependent phosphorylation of Cds1 at threonine-11 is required for Cds1 activation and function.
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PMID:Threonine-11, phosphorylated by Rad3 and atm in vitro, is required for activation of fission yeast checkpoint kinase Cds1. 1131 65

Genome integrity is monitored by a checkpoint that delays mitosis in response to DNA damage. This checkpoint is enforced by Chk1, a protein kinase that inhibits the mitotic inducer Cdc25. In fission yeast, Chk1 is regulated by a group of proteins that includes Rad3, a protein kinase related to human ATM and ATR. These kinases phosphorylate serine or threonine followed by glutamine (SQ/TQ). Fission yeast and human Chk1 proteins share two conserved SQ motifs at serine-345 and serine-367. Serine-345 of human Chk1 is phosphorylated in response to DNA damage. Here we report that Rad3 and ATM phosphorylate serine-345 of fission yeast Chk1. Mutation of serine-345 (chk1-S345A) abrogates Rad3-dependent phosphorylation of Chk1 in vivo. The chk1-S345A cells are sensitive to DNA damage and are checkpoint defective. In contrast, mutations of serine-367 and other SQ/TQ sites do not substantially impair the checkpoint or cause damage sensitivity. These findings attest to the importance of serine-345 phosphorylation for Chk1 function and strengthen evidence that transduction of the DNA damage checkpoint signal requires direct phosphorylation of Chk1 by Rad3.
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PMID:Serine-345 is required for Rad3-dependent phosphorylation and function of checkpoint kinase Chk1 in fission yeast. 1155 81

Jak2 is a member of the Janus family of tyrosine kinases and is known to be activated by a wide variety of ligands. Here, we sought to identify amino acid residues within Jak2 that are essential for its activation. We provide evidence that glutamic acid 1046 (E1046) is one such residue. Using molecular modeling algorithms of the Jak2 kinase domain, we identified a putative molecular interaction between E1046 and tryptophan 1020 (W1020). Conversion of E1046 to either arginine (E 1046R), alanine (E1046A), aspartic acid (E1046D) or glutamine (E1046Q) abolished Jak2 kinase activity as measured by autophosphorylation assays. Conversion of W1020 to glycine (W1020G) similarly abolished Jak2 kinase activity. Finally, we tested the ability of the E1046R mutant to activate the Jak/STAT signaling pathway in a ligand-dependent signaling system. The ability of angiotensin II to activate the Jak/STAT signaling pathway in cells expressing the E1046R mutant was severely compromised as measured by reduced (1) Jak2 autophosphorylation (2) Jak2 kinase activity (3) AT1/Jak2 co-association (4) Stat1 tyrosine phosphorylation and (5) angiotensin Il-mediated gene transcription. Thus, these studies demonstrate for the first time, the critical role of E1046 in mediating Jak2 activation and its subsequent downstream signaling events.
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PMID:Mutation of glutamic acid residue 1046 abolishes Jak2 tyrosine kinase activity. 1248 29

The checkpoint kinase Cds1 (Chk2) plays a key role in cell cycle checkpoint responses with functions in cell cycle arrest, DNA repair, and induction of apoptosis. Proper regulation of Cds1 is essential for appropriate cellular responses to checkpoint-inducing insults. While the kinase ATM has been shown to be important in the regulation of human Cds1 (hCds1), here we report that the kinases ATR and DNA-dependent protein kinase (DNA-PK) play more significant roles in the regulation of Xenopus Cds1 (XCds1). Under normal cell cycle conditions, nonactivated XCds1 constitutively associates with a Xenopus ATR complex. The association of XCds1 with this complex does not require a functional forkhead activation domain but does require a putative SH3 binding region that is found in XCds1. In response to double-stranded DNA ends, the amino terminus of XCds1 is rapidly phosphorylated in a sequential pattern. First DNA-PK phosphorylates serine 39, a site not previously recognized as important in Cds1 regulation. Xenopus ATM, ATR, and/or DNA-PK then phosphorylate three consensus serine/glutamine sites. Together, these phosphorylations have the dual function of inducing dissociation from the ATR complex and independently promoting the full activation of XCds1. Thus, the checkpoint-mediated activation of XCds1 requires phosphorylation by multiple phosphoinositide 3-kinase-related kinases, protein-protein dissociation, and autophosphorylation.
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PMID:Xenopus Cds1 is regulated by DNA-dependent protein kinase and ATR during the cell cycle checkpoint response to double-stranded DNA ends. 1550 99

H2AX is a core histone H2A variant that contains an absolutely conserved serine/glutamine (SQ) motif within an extended carboxy-terminal tail. H2AX phosphorylation at the SQ motif (gamma-H2AX) has been shown to increase dramatically upon exogenously introduced DNA double-strand breaks (DSBs). In this study, we use quantitative in situ approaches to investigate the spatial patterning and cell cycle dynamics of gamma-H2AX in a panel of normally growing (unirradiated) mammalian cell lines and cultures. We provide the first evidence for the existence of two distinct yet highly discernible gamma-H2AX focal populations: a small population of large amorphous foci that colocalize with numerous DNA DSB repair proteins and previously undescribed but much more abundant small foci. These small foci do not recruit proteins involved in DNA DSB repair. Cell cycle analyses reveal unexpected dynamics for gamma-H2AX in unirradiated mammalian cells that include an ATM-dependent phosphorylation that is maximal during M phase. Based upon similarities drawn from other histone posttranslational modifications and previous observations in haplo-insufficient (H2AX-/+) and null mice (H2AX-/-), gamma-H2AX may contribute to the fidelity of the mitotic process, even in the absence of DNA damage, thereby ensuring the faithful transmission of genetic information from one generation to the next.
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PMID:ATM-dependent DNA damage-independent mitotic phosphorylation of H2AX in normally growing mammalian cells. 1603 Feb 61


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