Gordon Gill - Academia.edu (original) (raw)

Papers by Gordon Gill

Research paper thumbnail of Protein modulation of cyclic nucleotide-dependent protein kinases

Methods in Enzymology, Feb 1, 1983

Research paper thumbnail of Identification of phosphorylation sites: use of the epidermal growth factor receptor

Methods in Enzymology, Feb 1, 1991

Research paper thumbnail of LIM domain recognition of a tyrosine-containing tight turn

The Journal of Biological Chemistry, Oct 7, 1994

sequence "codes" consisting of tight turn structures with an essential Tyr or Phe residue. To det... more sequence "codes" consisting of tight turn structures with an essential Tyr or Phe residue. To determine mechanisms through which cells recognize this information, we utilized exon 16 of the human insulin receptor in the two-hybrid system to isolate a novel 465-amino acid cytoplasmic protein that contains two LIM domains within its carboxyl terminus. Mutational analyses indicate that one of the Cys-rich Zna+ binding LIM domains specifically recognizes active but not inactive endocytic codes contained in exon 16. These findings suggest that LIM domain structures in proteins provide molecular recognition of Tyr-containing tight turn structures.

Research paper thumbnail of Ligand-Induced Endocytosis of Epidermal Growth Factor Receptors that are Defective in Binding Adaptor Proteins

Pnas, 1995

Ligand-activated epidermal growth factor receptors (EGFRs) associate with coated pit adaptor prot... more Ligand-activated epidermal growth factor receptors (EGFRs) associate with coated pit adaptor proteins (AP2) in vivo, implying a mechanism for receptor retention in coated pits during internalization. Using an in vitro binding assay, we localized the adaptor binding determinant to residues 970-991 of EGFRs and confirmed specificity by competition with a synthetic peptide corresponding to this sequence. A mutant EGFR lacking this AP2 binding determinant did not associate with AP2 in vivo but demonstrated internalization and down-regulation kinetics indistinguishable from its wild-type counterpart. Immunocytochemistry confirmed ligand-induced internalization of the mutant EGFR. These data suggest that endocytic determinants are distinct from AP2 binding determinants and that processes other than association with AP2 regulate endocytosis of EGFRs.

Research paper thumbnail of Hormonal regulation of the adrenocortical cell

Journal of supramolecular structure, 1980

Monolayer cultures of bovine and human adrenocortical cells have been used to study regulation of... more Monolayer cultures of bovine and human adrenocortical cells have been used to study regulation of growth and function. Homogeneous bovine adrenocortical cells exhibit a finite life span of approximately 60 generations in culture. Full maintenance of differentiated function (steroid hormone synthesis) requires an inducer such as ACTH and antioxidizing conditions. Full induction of differentiated function occurs only when cellular hypertrophy is stimulated by growth factors such as fibroblast growth factor and serum. ACTH and other agents that increase cellular cAMP inhibit replication but do not block growth factor-induced cellular hypertrophy. ACTH and growth factors together result in a hypertrophied, hyperfunctional cell. Replication ensues only when desensitization to the growth inhibitory effects of ACTH occurs. Cultures of the definitive and fetal zones of the human fetal adrenal cortex synthesize the steroids characteristic of the two zones in vivo. ACTH stimulates production ...

Research paper thumbnail of Relationship between production of epidermal growth factor receptors, gene amplification, and chromosome 7 translocation in variant A431 cells

Somatic cell and molecular genetics, 1985

Synthesis of the epidermal growth factor (EGF) receptor has been analyzed in a series of variant ... more Synthesis of the epidermal growth factor (EGF) receptor has been analyzed in a series of variant A431 human epidermoid carcinoma cell clones reported to contain different amounts of EGF binding sites. The amount of EGF receptor protein, quantitated by immunoaffinity chromatography, and EGF receptor mRNA, quantitated by cDNA hybridization, were closely correlated to the extent of EGF receptor gene amplification. This correlation existed in variants selected for reduced EGF receptors and in revertants from those variants with increased EGF receptors. There was also a correlation between the frequency of translocation of chromosome 7, containing the EGF receptor gene, and EGF receptor protein. These results support gene amplification as the mechanism enhancing A431 cell EGF receptor protein and determining growth responses.

Research paper thumbnail of Enhanced tumorigenesis of NR6 cells which express non-down-regulating epidermal growth factor receptors

Cancer research, Jan 15, 1991

Sequences in the regulatory carboxyl terminus of the epidermal growth factor (EGF) receptor are r... more Sequences in the regulatory carboxyl terminus of the epidermal growth factor (EGF) receptor are required for ligand-induced internalization via a high-affinity saturable endocytic pathway and for receptor down-regulation. To investigate the role of down-regulation in attenuating mitogenic signals, we compared the ability of NR6 cells expressing holo and mutant down-regulation defective EGF receptors to form tumors in athymic mice. NR6 cells expressing mutant EGF receptors reproducibly formed rapidly growing tumors, whereas cells expressing holo EGF receptors had a low tumorigenic potential. Serial passage of tumors of NR6 cells expressing mutant EGF receptors resulted in an enhanced rate of tumor formation that directly correlated with increased expression of mutant receptors. Tumor growth was inhibited by a competitive antagonist anti-EGF receptor monoclonal antibody. Excessive signaling from the cell surface can result from lack of sequences required for endocytosis and from satur...

Research paper thumbnail of Bio-molecular architects: a scaffold provided by the C-terminal domain of eukaryotic RNA polymerase II

Nano Reviews, 2010

In eukaryotic cells, the transcription of genes is accurately orchestrated both spatially and tem... more In eukaryotic cells, the transcription of genes is accurately orchestrated both spatially and temporally by the C-terminal domain of RNA polymerase II (CTD). The CTD provides a dynamic platform to recruit different regulators of the transcription apparatus. Different posttranslational modifications are precisely applied to specific sites of the CTD to coordinate transcription process. Regulators of the RNA polymerase II must identify specific sites in the CTD for cellular survival, metabolism, and development. Even though the CTD is disordered in the eukaryotic RNA polymerase II crystal structures due to its intrinsic flexibility, recent advances in the complex structural analysis of the CTD with its binding partners provide essential clues for understanding how selectivity is achieved for individual site recognition. The recent discoveries of the interactions between the CTD and histone modification enzymes disclose an important role of the CTD in epigenetic control of the eukaryotic gene expression. The intersection of the CTD code with the histone code discloses an intriguing yet complicated network for eukaryotic transcriptional regulation.

Research paper thumbnail of Regulation of the epidermal growth factor receptor by phosphorylation

Journal of Cellular Biochemistry, 1985

Administration of 17 beta-estradiol (E2) to immature female rats produces a 3-fold increase in 12... more Administration of 17 beta-estradiol (E2) to immature female rats produces a 3-fold increase in 125I-epidermal growth factor (EGF) binding to uterine membranes with no change in the affinity of membrane receptors for EGF. E2 treatment also increases the EGF receptor visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after affinity labeling of uterine membranes and the EGF-stimulated receptor autophosphorylation activity. In addition, E2 administration stimulates EGF-dependent tyrosine kinase activity in an assay system using exogenous angiotensin II as substrate. Following hormone treatment, EGF receptor levels increase between 6 and 12 h, remain elevated at 18 h, and decline between 24 and 36 h. This stimulation of EGF receptor levels by E2 is specific, since the non-estrogenic hormones progesterone, dexamethasone, and dihydrotestosterone fail to elevate receptor levels. E2-stimulated increases in EGF receptor levels are also blocked by cycloheximide and actinomycin D, suggesting that the observed effect represents de novo synthesis of the EGF receptor and may be mediated by a transcriptional mechanism. These results demonstrate that estrogen can regulate acutely the levels of EGF receptor in vivo and raise the possibility that events coupled to this receptor may play a role in estrogen-stimulated growth.

Research paper thumbnail of On the Mechanism of Action of ACTH

Proceedings of the 1970 Laurentian Hormone Conference, 1971

Research paper thumbnail of Supplemental Data Determinants for Dephosphorylation of the RNA Polymerase II C-Terminal Domain by Scp1

Research paper thumbnail of Regulation of cytokine-independent survival kinase (CISK) by the Phox homology domain and phosphoinositides

The Journal of Cell Biology, 2001

PKB/Akt and serum and glucocorticoid-regulated kinase (SGK) family kinases are important downstre... more PKB/Akt and serum and glucocorticoid-regulated kinase (SGK) family kinases are important downstream targets of phosphatidylinositol 3 (PI-3) kinase and have been shown to mediate a variety of cellular processes, including cell growth and survival. Although regulation of Akt can be achieved through several mechanisms, including its phosphoinositide-binding Pleckstrin homology (PH) domain, how SGK kinases are targeted and regulated remains to be elucidated. Unlike Akt, cytokine-independent survival kinase (CISK)/SGK3 contains a Phox homology (PX) domain. PX domains have been implicated in several cellular events involving membrane trafficking. However, their precise function remains unknown. We demonstrate here that the PX domain of CISK interacts with phosphatidylinositol (PtdIns)(3,5)P2, PtdIns(3,4,5)P3, and to a lesser extent PtdIns(4,5)P2. The CISK PX domain is required for targeting CISK to the endosomal compartment. Mutation in the PX domain that abolished its phospholipid binding ability not only disrupted CISK localization, but also resulted in a decrease in CISK activity in vivo. These results suggest that the PX domain regulates CISK localization and function through its direct interaction with phosphoinositides. Therefore, CISK and Akt have evolved to utilize different lipid binding domains to accomplish a similar mechanism of activation in response to PI-3 kinase signaling.

Research paper thumbnail of Ligand-induced transformation by a noninternalizing epidermal growth factor receptor

Science, 1990

Identification of a mutant epidermal growth factor (EGF) receptor that does not undergo downregul... more Identification of a mutant epidermal growth factor (EGF) receptor that does not undergo downregulation has provided a genetic probe to investigate the role of internalization in ligand-induced mitogenesis. Contact-inhibited cells expressing this internalization-defective receptor exhibited a normal mitogenic response at significantly lower ligand concentrations than did cells expressing wild-type receptors. A transformed phenotype and anchorage-independent growth were observed at ligand concentrations that failed to elicit these responses in cells expressing wild-type receptors. These findings imply that activation of the protein tyrosine kinase activity at the cell membrane is sufficient for the growth-enhancing effects of EGF. Thus, downregulation can serve as an attenuation mechanism, without which transformation ensues.

Research paper thumbnail of RGS-PX1, a GAP for Galpha s and Sorting Nexin in Vesicular Trafficking

Science, 2001

Heterotrimeric GTP-binding proteins (G proteins) control cellular functions by transducing signal... more Heterotrimeric GTP-binding proteins (G proteins) control cellular functions by transducing signals from the outside to the inside of cells. Regulator of G protein signaling (RGS) proteins are key modulators of the amplitude and duration of G protein-mediated signaling through their ability to serve as guanosine triphosphatase-activating proteins (GAPs). We have identified RGS-PX1, a Galpha(s)-specific GAP. The RGS domain of RGS-PX1 specifically interacted with Galpha(s), accelerated its GTP hydrolysis, and attenuated Galpha(s)-mediated signaling. RGS-PX1 also contains a Phox (PX) domain that resembles those in sorting nexin (SNX) proteins. Expression of RGS-PX1 delayed lysosomal degradation of the EGF receptor. Because of its bifunctional role as both a GAP and a SNX, RGS-PX1 may link heterotrimeric G protein signaling and vesicular trafficking.

Research paper thumbnail of Expression cloning of human EGF receptor complementary DNA: gene amplification and three related messenger RNA products in A431 cells

Science, 1984

In order to further define the mechanisms by which polypeptide growth factors regulate gene trans... more In order to further define the mechanisms by which polypeptide growth factors regulate gene transcription and cellular growth, expression cloning techniques were used to select human epidermal growth factor (EGF) receptor complementary DNA clones. The EGF 3' coding domain shows striking homology to the transforming gene product of avian erythroblastosis virus (v-erbB). Over-expression of EGF receptors in A431 cell lines correlates with increased EGF receptor mRNA levels and amplification (up to 110 times) of the apparently singular EGF receptor gene. There appear to be three cytoplasmic polyadenylated RNA products of EGF receptor gene expression in A431 cells, one of which contains only 5' (EGF binding domain) sequences and is postulated to encode the secreted EGF receptor-related protein.

Research paper thumbnail of Small CTD Phosphatases Function in Silencing Neuronal Gene Expression

Science, 2005

Neuronal gene transcription is repressed in non-neuronal cells by the repressor element 1 (RE-1)-... more Neuronal gene transcription is repressed in non-neuronal cells by the repressor element 1 (RE-1)-silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF) complex. To understand how this silencing is achieved, we examined a family of class-C RNA polymerase II (RNAPII) carboxyl-terminal domain (CTD) phosphatases [small CTD phosphatases (SCPs) 1 to 3], whose expression is restricted to non-neuronal tissues. We show that REST/NRSF recruits SCPs to neuronal genes that contain RE-1 elements, leading to neuronal gene silencing in non-neuronal cells. Phosphatase-inactive forms of SCP interfere with REST/NRSF function and promote neuronal differentiation of P19 stem cells. Likewise, small interfering RNA directed to the single Drosophila SCP unmasks neuronal gene expression in S2 cells. Thus, SCP activity is an evolutionarily conserved transcriptional regulator that acts globally to silence neuronal genes.

Research paper thumbnail of Production of an epidermal growth factor receptor-related protein

Science, 1984

Human epidermoid carcinoma A431 cells in culture produce a soluble 105-kilodalton protein which, ... more Human epidermoid carcinoma A431 cells in culture produce a soluble 105-kilodalton protein which, by the criteria of epidermal growth factor (EGF) binding, recognition by monoclonal and polyclonal antibodies to the EGF receptor, amino-terminal sequence analysis and carbohydrate content, is related to the cell surface domain of the EGF receptor. The high rate of production and the finding that with biosynthetic labeling the specific activity of this 105-kilodalton protein exceeds that of the intact receptor indicate that it is not derived from membrane-bound mature receptor but is separately produced by the cell. These cells thus separately synthesize an EGF receptor that is inserted into the membrane and an EGF receptor-related protein that is secreted.

Research paper thumbnail of The cAMP-regulated transcription factor CREB interacts with a component of the TFIID complex

Proceedings of the National Academy of Sciences, 1994

cAMP regulates the expression of a number of genes through the protein kinase A-mediated phosphor... more cAMP regulates the expression of a number of genes through the protein kinase A-mediated phosphorylation of CREB at Ser-133. The effects of Ser-133 phosphorylation appear to be primarily transmitted through a modulatory kinase-inducible domain in CREB that functions cooperatively with a 120-amino acid glutannine-rich region (Q2) to stimulate transcription. Indeed, tihe kinase-inducible domain activity alone is not sufficient to sustain a transcriptional response as illustrated by the CREM family of repressors, which contain the kinase-Inducible domain but lack the Q2 region. Here we demonstrate that Q2 functions as a potent constitutive activator in vitro. The transcription factor TFHD fraption supports transcriptional activation by Q2, although the "TATA" binding protein alone does not, suggesting that other components of the TFIID complex mediate the response to CREB Q2. In fact, Q2 associates with the TATA binding protein-associated factor

Research paper thumbnail of Endosomal localization and function of sorting nexin 1

Proceedings of the National Academy of Sciences, 2002

There are 17 human members of the sorting nexin (SNX) family of proteins that contain Phox (PX) d... more There are 17 human members of the sorting nexin (SNX) family of proteins that contain Phox (PX) domains. Yeast orthologs function in vesicular trafficking and mammalian proteins have been implicated in endocytic trafficking of cell surface receptors. The first member of this family, SNX1, was identified via interaction with the epidermal growth factor receptor. The present studies indicate that SNX1 and SNX2 are colocalized to tubulovesicular endosomal membranes and this localization depends on PI 3-kinase activity. Point mutations in the PX domain that abolish recognition of phosphorylated phosphatidylinositol (PtdIns) in vitro abolish vesicle localization in vivo indicating that lipid binding by the PX domain is necessary for localization to vesicle membranes. Deletion of a predicted coiled-coil region in the COOH terminus of SNX1 also abolished vesicle localization, indicating that this helical domain, too, is necessary for SNX1 localization. Thus, both PX domain recognition of PtdIns and COOH terminal helical domains are necessary for localization of SNX1 with neither alone being sufficient. Regulated overexpression of the NH(2) terminus of SNX1 containing the PX domain decreased the rate of ligand-induced epidermal growth factor receptor degradation, an effect consistent with inhibition of endogenous SNX1 function in the endosome compartment. SNX1 thus functions in regulating trafficking in the endosome compartment via PX domain recognition of phosphorylated PtdIns and via interaction with other protein components.

Research paper thumbnail of Purification and characterization of 3':5'-cyclic GMP-dependent protein kinase

Proceedings of the National Academy of Sciences, 1976

Guanosine 3':5'-cyclic monophosphate (cGMP)dependent protein kinase has been purified to homogene... more Guanosine 3':5'-cyclic monophosphate (cGMP)dependent protein kinase has been purified to homogeneity from bovine lung by affinity chromatography and characterized. Partially purified protein kinase, specifically activated by low concentrations of cGMP (22 nM), was adsorbed onto 8(2-aminoethyl)amino-adenosine 3':5'-cyclic monophosphate-Sepharose. After washing to remove nonspeic proteins,

Research paper thumbnail of Protein modulation of cyclic nucleotide-dependent protein kinases

Methods in Enzymology, Feb 1, 1983

Research paper thumbnail of Identification of phosphorylation sites: use of the epidermal growth factor receptor

Methods in Enzymology, Feb 1, 1991

Research paper thumbnail of LIM domain recognition of a tyrosine-containing tight turn

The Journal of Biological Chemistry, Oct 7, 1994

sequence "codes" consisting of tight turn structures with an essential Tyr or Phe residue. To det... more sequence "codes" consisting of tight turn structures with an essential Tyr or Phe residue. To determine mechanisms through which cells recognize this information, we utilized exon 16 of the human insulin receptor in the two-hybrid system to isolate a novel 465-amino acid cytoplasmic protein that contains two LIM domains within its carboxyl terminus. Mutational analyses indicate that one of the Cys-rich Zna+ binding LIM domains specifically recognizes active but not inactive endocytic codes contained in exon 16. These findings suggest that LIM domain structures in proteins provide molecular recognition of Tyr-containing tight turn structures.

Research paper thumbnail of Ligand-Induced Endocytosis of Epidermal Growth Factor Receptors that are Defective in Binding Adaptor Proteins

Pnas, 1995

Ligand-activated epidermal growth factor receptors (EGFRs) associate with coated pit adaptor prot... more Ligand-activated epidermal growth factor receptors (EGFRs) associate with coated pit adaptor proteins (AP2) in vivo, implying a mechanism for receptor retention in coated pits during internalization. Using an in vitro binding assay, we localized the adaptor binding determinant to residues 970-991 of EGFRs and confirmed specificity by competition with a synthetic peptide corresponding to this sequence. A mutant EGFR lacking this AP2 binding determinant did not associate with AP2 in vivo but demonstrated internalization and down-regulation kinetics indistinguishable from its wild-type counterpart. Immunocytochemistry confirmed ligand-induced internalization of the mutant EGFR. These data suggest that endocytic determinants are distinct from AP2 binding determinants and that processes other than association with AP2 regulate endocytosis of EGFRs.

Research paper thumbnail of Hormonal regulation of the adrenocortical cell

Journal of supramolecular structure, 1980

Monolayer cultures of bovine and human adrenocortical cells have been used to study regulation of... more Monolayer cultures of bovine and human adrenocortical cells have been used to study regulation of growth and function. Homogeneous bovine adrenocortical cells exhibit a finite life span of approximately 60 generations in culture. Full maintenance of differentiated function (steroid hormone synthesis) requires an inducer such as ACTH and antioxidizing conditions. Full induction of differentiated function occurs only when cellular hypertrophy is stimulated by growth factors such as fibroblast growth factor and serum. ACTH and other agents that increase cellular cAMP inhibit replication but do not block growth factor-induced cellular hypertrophy. ACTH and growth factors together result in a hypertrophied, hyperfunctional cell. Replication ensues only when desensitization to the growth inhibitory effects of ACTH occurs. Cultures of the definitive and fetal zones of the human fetal adrenal cortex synthesize the steroids characteristic of the two zones in vivo. ACTH stimulates production ...

Research paper thumbnail of Relationship between production of epidermal growth factor receptors, gene amplification, and chromosome 7 translocation in variant A431 cells

Somatic cell and molecular genetics, 1985

Synthesis of the epidermal growth factor (EGF) receptor has been analyzed in a series of variant ... more Synthesis of the epidermal growth factor (EGF) receptor has been analyzed in a series of variant A431 human epidermoid carcinoma cell clones reported to contain different amounts of EGF binding sites. The amount of EGF receptor protein, quantitated by immunoaffinity chromatography, and EGF receptor mRNA, quantitated by cDNA hybridization, were closely correlated to the extent of EGF receptor gene amplification. This correlation existed in variants selected for reduced EGF receptors and in revertants from those variants with increased EGF receptors. There was also a correlation between the frequency of translocation of chromosome 7, containing the EGF receptor gene, and EGF receptor protein. These results support gene amplification as the mechanism enhancing A431 cell EGF receptor protein and determining growth responses.

Research paper thumbnail of Enhanced tumorigenesis of NR6 cells which express non-down-regulating epidermal growth factor receptors

Cancer research, Jan 15, 1991

Sequences in the regulatory carboxyl terminus of the epidermal growth factor (EGF) receptor are r... more Sequences in the regulatory carboxyl terminus of the epidermal growth factor (EGF) receptor are required for ligand-induced internalization via a high-affinity saturable endocytic pathway and for receptor down-regulation. To investigate the role of down-regulation in attenuating mitogenic signals, we compared the ability of NR6 cells expressing holo and mutant down-regulation defective EGF receptors to form tumors in athymic mice. NR6 cells expressing mutant EGF receptors reproducibly formed rapidly growing tumors, whereas cells expressing holo EGF receptors had a low tumorigenic potential. Serial passage of tumors of NR6 cells expressing mutant EGF receptors resulted in an enhanced rate of tumor formation that directly correlated with increased expression of mutant receptors. Tumor growth was inhibited by a competitive antagonist anti-EGF receptor monoclonal antibody. Excessive signaling from the cell surface can result from lack of sequences required for endocytosis and from satur...

Research paper thumbnail of Bio-molecular architects: a scaffold provided by the C-terminal domain of eukaryotic RNA polymerase II

Nano Reviews, 2010

In eukaryotic cells, the transcription of genes is accurately orchestrated both spatially and tem... more In eukaryotic cells, the transcription of genes is accurately orchestrated both spatially and temporally by the C-terminal domain of RNA polymerase II (CTD). The CTD provides a dynamic platform to recruit different regulators of the transcription apparatus. Different posttranslational modifications are precisely applied to specific sites of the CTD to coordinate transcription process. Regulators of the RNA polymerase II must identify specific sites in the CTD for cellular survival, metabolism, and development. Even though the CTD is disordered in the eukaryotic RNA polymerase II crystal structures due to its intrinsic flexibility, recent advances in the complex structural analysis of the CTD with its binding partners provide essential clues for understanding how selectivity is achieved for individual site recognition. The recent discoveries of the interactions between the CTD and histone modification enzymes disclose an important role of the CTD in epigenetic control of the eukaryotic gene expression. The intersection of the CTD code with the histone code discloses an intriguing yet complicated network for eukaryotic transcriptional regulation.

Research paper thumbnail of Regulation of the epidermal growth factor receptor by phosphorylation

Journal of Cellular Biochemistry, 1985

Administration of 17 beta-estradiol (E2) to immature female rats produces a 3-fold increase in 12... more Administration of 17 beta-estradiol (E2) to immature female rats produces a 3-fold increase in 125I-epidermal growth factor (EGF) binding to uterine membranes with no change in the affinity of membrane receptors for EGF. E2 treatment also increases the EGF receptor visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after affinity labeling of uterine membranes and the EGF-stimulated receptor autophosphorylation activity. In addition, E2 administration stimulates EGF-dependent tyrosine kinase activity in an assay system using exogenous angiotensin II as substrate. Following hormone treatment, EGF receptor levels increase between 6 and 12 h, remain elevated at 18 h, and decline between 24 and 36 h. This stimulation of EGF receptor levels by E2 is specific, since the non-estrogenic hormones progesterone, dexamethasone, and dihydrotestosterone fail to elevate receptor levels. E2-stimulated increases in EGF receptor levels are also blocked by cycloheximide and actinomycin D, suggesting that the observed effect represents de novo synthesis of the EGF receptor and may be mediated by a transcriptional mechanism. These results demonstrate that estrogen can regulate acutely the levels of EGF receptor in vivo and raise the possibility that events coupled to this receptor may play a role in estrogen-stimulated growth.

Research paper thumbnail of On the Mechanism of Action of ACTH

Proceedings of the 1970 Laurentian Hormone Conference, 1971

Research paper thumbnail of Supplemental Data Determinants for Dephosphorylation of the RNA Polymerase II C-Terminal Domain by Scp1

Research paper thumbnail of Regulation of cytokine-independent survival kinase (CISK) by the Phox homology domain and phosphoinositides

The Journal of Cell Biology, 2001

PKB/Akt and serum and glucocorticoid-regulated kinase (SGK) family kinases are important downstre... more PKB/Akt and serum and glucocorticoid-regulated kinase (SGK) family kinases are important downstream targets of phosphatidylinositol 3 (PI-3) kinase and have been shown to mediate a variety of cellular processes, including cell growth and survival. Although regulation of Akt can be achieved through several mechanisms, including its phosphoinositide-binding Pleckstrin homology (PH) domain, how SGK kinases are targeted and regulated remains to be elucidated. Unlike Akt, cytokine-independent survival kinase (CISK)/SGK3 contains a Phox homology (PX) domain. PX domains have been implicated in several cellular events involving membrane trafficking. However, their precise function remains unknown. We demonstrate here that the PX domain of CISK interacts with phosphatidylinositol (PtdIns)(3,5)P2, PtdIns(3,4,5)P3, and to a lesser extent PtdIns(4,5)P2. The CISK PX domain is required for targeting CISK to the endosomal compartment. Mutation in the PX domain that abolished its phospholipid binding ability not only disrupted CISK localization, but also resulted in a decrease in CISK activity in vivo. These results suggest that the PX domain regulates CISK localization and function through its direct interaction with phosphoinositides. Therefore, CISK and Akt have evolved to utilize different lipid binding domains to accomplish a similar mechanism of activation in response to PI-3 kinase signaling.

Research paper thumbnail of Ligand-induced transformation by a noninternalizing epidermal growth factor receptor

Science, 1990

Identification of a mutant epidermal growth factor (EGF) receptor that does not undergo downregul... more Identification of a mutant epidermal growth factor (EGF) receptor that does not undergo downregulation has provided a genetic probe to investigate the role of internalization in ligand-induced mitogenesis. Contact-inhibited cells expressing this internalization-defective receptor exhibited a normal mitogenic response at significantly lower ligand concentrations than did cells expressing wild-type receptors. A transformed phenotype and anchorage-independent growth were observed at ligand concentrations that failed to elicit these responses in cells expressing wild-type receptors. These findings imply that activation of the protein tyrosine kinase activity at the cell membrane is sufficient for the growth-enhancing effects of EGF. Thus, downregulation can serve as an attenuation mechanism, without which transformation ensues.

Research paper thumbnail of RGS-PX1, a GAP for Galpha s and Sorting Nexin in Vesicular Trafficking

Science, 2001

Heterotrimeric GTP-binding proteins (G proteins) control cellular functions by transducing signal... more Heterotrimeric GTP-binding proteins (G proteins) control cellular functions by transducing signals from the outside to the inside of cells. Regulator of G protein signaling (RGS) proteins are key modulators of the amplitude and duration of G protein-mediated signaling through their ability to serve as guanosine triphosphatase-activating proteins (GAPs). We have identified RGS-PX1, a Galpha(s)-specific GAP. The RGS domain of RGS-PX1 specifically interacted with Galpha(s), accelerated its GTP hydrolysis, and attenuated Galpha(s)-mediated signaling. RGS-PX1 also contains a Phox (PX) domain that resembles those in sorting nexin (SNX) proteins. Expression of RGS-PX1 delayed lysosomal degradation of the EGF receptor. Because of its bifunctional role as both a GAP and a SNX, RGS-PX1 may link heterotrimeric G protein signaling and vesicular trafficking.

Research paper thumbnail of Expression cloning of human EGF receptor complementary DNA: gene amplification and three related messenger RNA products in A431 cells

Science, 1984

In order to further define the mechanisms by which polypeptide growth factors regulate gene trans... more In order to further define the mechanisms by which polypeptide growth factors regulate gene transcription and cellular growth, expression cloning techniques were used to select human epidermal growth factor (EGF) receptor complementary DNA clones. The EGF 3' coding domain shows striking homology to the transforming gene product of avian erythroblastosis virus (v-erbB). Over-expression of EGF receptors in A431 cell lines correlates with increased EGF receptor mRNA levels and amplification (up to 110 times) of the apparently singular EGF receptor gene. There appear to be three cytoplasmic polyadenylated RNA products of EGF receptor gene expression in A431 cells, one of which contains only 5' (EGF binding domain) sequences and is postulated to encode the secreted EGF receptor-related protein.

Research paper thumbnail of Small CTD Phosphatases Function in Silencing Neuronal Gene Expression

Science, 2005

Neuronal gene transcription is repressed in non-neuronal cells by the repressor element 1 (RE-1)-... more Neuronal gene transcription is repressed in non-neuronal cells by the repressor element 1 (RE-1)-silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF) complex. To understand how this silencing is achieved, we examined a family of class-C RNA polymerase II (RNAPII) carboxyl-terminal domain (CTD) phosphatases [small CTD phosphatases (SCPs) 1 to 3], whose expression is restricted to non-neuronal tissues. We show that REST/NRSF recruits SCPs to neuronal genes that contain RE-1 elements, leading to neuronal gene silencing in non-neuronal cells. Phosphatase-inactive forms of SCP interfere with REST/NRSF function and promote neuronal differentiation of P19 stem cells. Likewise, small interfering RNA directed to the single Drosophila SCP unmasks neuronal gene expression in S2 cells. Thus, SCP activity is an evolutionarily conserved transcriptional regulator that acts globally to silence neuronal genes.

Research paper thumbnail of Production of an epidermal growth factor receptor-related protein

Science, 1984

Human epidermoid carcinoma A431 cells in culture produce a soluble 105-kilodalton protein which, ... more Human epidermoid carcinoma A431 cells in culture produce a soluble 105-kilodalton protein which, by the criteria of epidermal growth factor (EGF) binding, recognition by monoclonal and polyclonal antibodies to the EGF receptor, amino-terminal sequence analysis and carbohydrate content, is related to the cell surface domain of the EGF receptor. The high rate of production and the finding that with biosynthetic labeling the specific activity of this 105-kilodalton protein exceeds that of the intact receptor indicate that it is not derived from membrane-bound mature receptor but is separately produced by the cell. These cells thus separately synthesize an EGF receptor that is inserted into the membrane and an EGF receptor-related protein that is secreted.

Research paper thumbnail of The cAMP-regulated transcription factor CREB interacts with a component of the TFIID complex

Proceedings of the National Academy of Sciences, 1994

cAMP regulates the expression of a number of genes through the protein kinase A-mediated phosphor... more cAMP regulates the expression of a number of genes through the protein kinase A-mediated phosphorylation of CREB at Ser-133. The effects of Ser-133 phosphorylation appear to be primarily transmitted through a modulatory kinase-inducible domain in CREB that functions cooperatively with a 120-amino acid glutannine-rich region (Q2) to stimulate transcription. Indeed, tihe kinase-inducible domain activity alone is not sufficient to sustain a transcriptional response as illustrated by the CREM family of repressors, which contain the kinase-Inducible domain but lack the Q2 region. Here we demonstrate that Q2 functions as a potent constitutive activator in vitro. The transcription factor TFHD fraption supports transcriptional activation by Q2, although the "TATA" binding protein alone does not, suggesting that other components of the TFIID complex mediate the response to CREB Q2. In fact, Q2 associates with the TATA binding protein-associated factor

Research paper thumbnail of Endosomal localization and function of sorting nexin 1

Proceedings of the National Academy of Sciences, 2002

There are 17 human members of the sorting nexin (SNX) family of proteins that contain Phox (PX) d... more There are 17 human members of the sorting nexin (SNX) family of proteins that contain Phox (PX) domains. Yeast orthologs function in vesicular trafficking and mammalian proteins have been implicated in endocytic trafficking of cell surface receptors. The first member of this family, SNX1, was identified via interaction with the epidermal growth factor receptor. The present studies indicate that SNX1 and SNX2 are colocalized to tubulovesicular endosomal membranes and this localization depends on PI 3-kinase activity. Point mutations in the PX domain that abolish recognition of phosphorylated phosphatidylinositol (PtdIns) in vitro abolish vesicle localization in vivo indicating that lipid binding by the PX domain is necessary for localization to vesicle membranes. Deletion of a predicted coiled-coil region in the COOH terminus of SNX1 also abolished vesicle localization, indicating that this helical domain, too, is necessary for SNX1 localization. Thus, both PX domain recognition of PtdIns and COOH terminal helical domains are necessary for localization of SNX1 with neither alone being sufficient. Regulated overexpression of the NH(2) terminus of SNX1 containing the PX domain decreased the rate of ligand-induced epidermal growth factor receptor degradation, an effect consistent with inhibition of endogenous SNX1 function in the endosome compartment. SNX1 thus functions in regulating trafficking in the endosome compartment via PX domain recognition of phosphorylated PtdIns and via interaction with other protein components.

Research paper thumbnail of Purification and characterization of 3':5'-cyclic GMP-dependent protein kinase

Proceedings of the National Academy of Sciences, 1976

Guanosine 3':5'-cyclic monophosphate (cGMP)dependent protein kinase has been purified to homogene... more Guanosine 3':5'-cyclic monophosphate (cGMP)dependent protein kinase has been purified to homogeneity from bovine lung by affinity chromatography and characterized. Partially purified protein kinase, specifically activated by low concentrations of cGMP (22 nM), was adsorbed onto 8(2-aminoethyl)amino-adenosine 3':5'-cyclic monophosphate-Sepharose. After washing to remove nonspeic proteins,