Direct imaging of phosphorylation‐dependent conformational change and DNA binding of CREB by electron microscopy

Usukura, Jiro; Shimomura, Atsushi; Kobayashi, Kazuto; Nagatsu, Toshiharu; Hagiwara, Masatoshi

doi:10.1046/j.1365-2443.2000.00345.x

Cited by 12 publications

(10 citation statements)

References 22 publications

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“…However, electrophoretic mobility shift assays (EMSAs) demonstrated that the pCREB/DNA complex migrates with reduced mobility relative to CREB/DNA (Fig. 6A), consistent with previous studies (22,57). This data suggests that phosphorylation induces a structural change that increases the apparent size or shape of the CREB/DNA complex.…”

Section: Dna Binding and Phosphorylation Bothsupporting

confidence: 91%

“…Although both CREB and pCREB bound to DNA elute from the gel filtration column at an unusually large apparent molecular weight, perhaps reflecting an extraordinarily asymmetric or oblong structure, a further increase in apparent size was observed with the phosphorylated complex. In support of these observations, visualization of CREB dimers by electron microscopy also suggests an increase in the area between the two monomers upon phosphorylation (57). Similarly, Ferguson plot analysis has shown an increase in the spherical size and net positive surface charge of the CREB/DNA complex upon phosphorylation (29).…”

Section: Discussionmentioning

confidence: 79%

“…We found that DNA-bound pCREB/bZIP heterodimers exhibit equivalent protection from PP1 dephosphorylation as full-length homodimers, and both display a comparable, less compact tertiary structure than that of unphosphorylated CREB complexes. The notion that the intrinsic structure of one monomer in the DNA-bound pCREB dimer is independent of intermolecular interactions with its partner is supported by structural studies showing a single phosphorylated KID interacts with a single KIX (24,57). Furthermore, deletion of basic amino acids near Ser 133 in CREB 341 ⌬121-125, which resulted in a less compact tertiary structure like that exhibited by pCREB, suggests that the negative charges from the phosphate group disrupt intramolecular electrostatic interactions between these basic residues and neighboring acidic residues.…”

Section: Discussionmentioning

confidence: 99%

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DNA Binding and Phosphorylation Induce Conformational Alterations in the Kinase-inducible Domain of CREB

Sharma

Lopez

Nyborg

2007

Journal of Biological Chemistry

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CREB-mediated activation of target gene transcription is stimulated by protein kinase A (PKA) phosphorylation at serine 133. This is followed by recruitment of the coactivators CREB-binding protein (CBP) or p300. Conversely, the decline in expression during the attenuation phase is linked to CREB dephosphorylation by nuclear phosphatases. The CREB bZIP domain, which promotes dimerization and promoter binding, as well as the kinase-inducible domain (KID), which interacts with the KIX domain of CBP/p300, are both largely unstructured in solution and become more structured once bound to their respective ligands. In this study, we biochemically characterize DNA-and phosphorylation-induced conformational alterations in CREB that may play a role in its transcriptionally poised, activated state. We find that sequencespecific DNA binding of pCREB renders the protein resistant to serine 133 dephosphorylation by protein phosphatase 1. Paradoxically, CREB bound to DNA and chromatin is efficiently phosphorylated by PKA, indicating that the KID region exists in a different conformation depending on its phosphorylation state. Consistent with this observation, we find that phosphorylation of DNA-bound CREB promotes an alternate conformation characterized by an apparent increase in the size or asymmetry of the complex and a qualitative change in proteolytic sensitivity. Together, our data indicate that DNA binding promotes a global conformational change in CREB that alters the structure of KID. PKA phosphorylation of KID in the DNA-bound state induces a phosphatase-resistant conformation that may prolong transcriptional activity.The cyclic AMP response element-binding protein (CREB) 2 induces expression of many cellular genes in response to phosphorylation by several kinases including the cAMP-dependent kinase (PKA) (1). CREB binds via its basic leucine zipper (bZIP) domain as a dimer to cAMP response elements (CREs) containing the 5Ј-TGACGTCA-3Ј consensus motif. CREB has been shown to be constitutively bound to promoters carrying CREs in vivo (2-4). Activation of transcription by CREB occurs cooperatively via the kinase-inducible domain (KID) and the constitutive glutamine-rich domain (Q2) (5-7). Following PKA phosphorylation at serine 133 (Ser 133 ) of KID, CREB recruits CREB-binding protein (CBP) or its paralogue, p300 (8, 9). CREB-mediated activation of transcription follows "burstattenuation" kinetics and maximal transcription coincides with maximal levels of phosphorylation (10, 11). Two major serine/ threonine protein phosphatases, PP1 and PP2A, have been implicated as the enzymes responsible for CREB dephosphorylation and transcriptional attenuation (10, 12, 13).Many transcription factors are thought to undergo induced conformational changes in a ligand-dependent fashion. DNAinduced conformational changes have been described for several bZIP family proteins, including CREB, GCN5, C/EBP, c-Jun, and c-Fos (14 -16). In CREB, the DNA-binding bZIP domain displays a 20% increase in helical structure following sequence-specifi...

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Section: Dna Binding and Phosphorylation Bothsupporting

confidence: 91%

Section: Discussionmentioning

confidence: 79%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

DNA Binding and Phosphorylation Induce Conformational Alterations in the Kinase-inducible Domain of CREB

Sharma

Lopez

Nyborg

2007

Journal of Biological Chemistry

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“…It is unclear whether phosphorylation causes a conformational change in Sp1 or the promoter it binds 51 or if its modification engages and facilitates assembly of a transcriptional cofactor complex 52 favoring increased Sp1-dependent gene expression. Lessons in subsequent studies may be drawn from other transcription factors.…”

Section: Discussionmentioning

confidence: 99%

Angiotensin II–Inducible Platelet-Derived Growth Factor-D Transcription Requires Specific Ser/Thr Residues in the Second Zinc Finger Region of Sp1

Tan

Midgley

Kavurma

et al. 2008

Circulation Research

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Abstract-Sp1, the first identified and cloned transcription factor, regulates gene expression via multiple mechanisms including direct protein-DNA interactions, protein-protein interactions, chromatin remodeling, and maintenance of methylation-free CpG islands. Sp1 is itself regulated at different levels, for example, by glycosylation, acetylation, and phosphorylation by kinases such as the atypical protein kinase C-. Although Sp1 controls the basal and inducible regulation of many genes, the posttranslational processes regulating its function and their relevance to pathology are not well understood. Here we have used a variety of approaches to identify 3 amino acids (Thr668, Ser670, and Thr681) in the zinc finger domain of Sp1 that are modified by PKC-and have generated novel anti-peptide antibodies recognizing the PKC--phosphorylated form of Sp1. Angiotensin II, which activates PKC-phosphorylation (at Thr410) via the angiotensin II type 1 receptor, stimulates Sp1 phosphorylation and increases Sp1 binding to the platelet-derived growth factor-D promoter. All 3 residues in Sp1 (Thr668, Ser670, and Thr681) are required for Sp1-dependent platelet-derived growth factor-D activation in response to angiotensin II. Immunohistochemical analysis revealed that phosphorylated Sp1 is expressed in smooth muscle cells of human atherosclerotic plaques and is dynamically expressed together with platelet-derived growth factor-D in smooth muscle cells of the injured rat carotid artery wall. This study provides new insights into the regulatory mechanisms controlling the PKC--phospho-Sp1 axis and angiotensin II-inducible gene expression. (Circ Res. 2008;102:e38-e51.)

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“…The transcriptional regulation of the TH gene has been extensively studied, and many transcription factors were suggested to regulate TH gene expression. CREB, ATF-1, and CREM were shown to recognize the cAMP-responsive element (CRE) located at position Ϫ45/Ϫ38 in the TH promoter (2)(3)(4)(5)(6). CREB was reported to mediate basal and cAMP-induced TH transcription in various cultured cells including PC12 cells by the use of dominant-negative CREB protein and antisense RNA against CREB (7)(8)(9)(10).…”

mentioning

confidence: 99%

Identification of ATF-2 as a Transcriptional Regulator for the Tyrosine Hydroxylase Gene

Suzuki

Yamakuni

Hagiwara

et al. 2002

Journal of Biological Chemistry

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Transcriptional regulation of catecholamine-synthesizing genes is important for the determination of neurotransmitters during brain development. We found that three catecholamine-synthesizing genes were transcriptionally up-regulated in cloned PC12D cells overexpressing V-1, a protein that is highly expressed during postnatal brain development (1). To reveal the molecular mechanism to regulate the expression of tyrosine hydroxylase (TH), which is the rate-limiting enzyme for catecholamine biosynthesis, we analyzed the transcription factors responsible for TH induction in the V-1 clonal cells. First, by using reporter constructs, we found that the transcription mediated by cAMP-responsive element (CRE) was selectively enhanced in the V-1 cells, and TH promoter activity was totally dependent on the CRE in the promoter region of the TH gene. Next, immunoblot analyses and a transactivation assay using a GAL4 reporter system revealed that ATF-2, but not cAMP-responsive element-binding protein (CREB), was highly phosphorylated and activated in the V-1 cells, while both CREB and ATF-2 were bound to the TH-CRE. Finally, the enhanced TH promoter activity was competitively attenuated by expression of a plasmid containing the ATF-2 transactivation domain. These data demonstrated that activation of ATF-2 resulted in the increased transcription of the TH gene and suggest that ATF-2 may be deeply involved in the transcriptional regulation of catecholamine-synthesizing genes during neural development.Catecholamines are synthesized from L-tyrosine by the sequential action of four enzymes: tyrosine is converted to DOPA by tyrosine hydroxylase (TH), 1 DOPA to dopamine by aromatic L-amino acid decarboxylase (AADC), dopamine to norepinephrine by dopamine ␤-hydroxylase (DBH), and norepinephrine to epinephrine by phenylethanolamine N-methyltransferase. The regulation of the gene expression of these enzymes is important for the determination of the expression of neurotransmitters during brain development as well as for brain function under physiological and pathological conditions. TH is the rate-limiting enzyme for catecholamine biosynthesis. The transcriptional regulation of the TH gene has been extensively studied, and many transcription factors were suggested to regulate TH gene expression. CREB, ATF-1, and CREM were shown to recognize the cAMP-responsive element (CRE) located at position Ϫ45/Ϫ38 in the TH promoter (2-6). CREB was reported to mediate basal and cAMP-induced TH transcription in various cultured cells including PC12 cells by the use of dominant-negative CREB protein and antisense RNA against CREB (7-10). CREB is activated by phosphorylation on Ser 133 (11,12). The activation of CREB by phosphorylation has been shown to mediate PKA-dependent (7, 13-15) and -independent (13, 14) induction of TH transcription. Functional and physiological roles of ATF-1 and CREM for TH transcription remained unclear. AP-1 transcription factors bound to the TPA-responsive element located at Ϫ205/Ϫ199 in the TH promoter, which also plays ...

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Direct imaging of phosphorylation‐dependent conformational change and DNA binding of CREB by electron microscopy

Cited by 12 publications

References 22 publications

DNA Binding and Phosphorylation Induce Conformational Alterations in the Kinase-inducible Domain of CREB

DNA Binding and Phosphorylation Induce Conformational Alterations in the Kinase-inducible Domain of CREB

Angiotensin II–Inducible Platelet-Derived Growth Factor-D Transcription Requires Specific Ser/Thr Residues in the Second Zinc Finger Region of Sp1

Identification of ATF-2 as a Transcriptional Regulator for the Tyrosine Hydroxylase Gene

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