A novel target recognition revealed by calmodulin in complex with the basic helix–loop–helix transcription factor SEF2‐1/E2‐2

Larsson, Göran; Schleucher, Jürgen; Onions, Jacqueline; Hermann, Stefan; Grundström, Thoman; Wijmenga, Sybren S.

doi:10.1110/ps.28401

Cited by 21 publications

(25 citation statements)

References 55 publications

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“…Different from these previously reported structures, CaM in the CaM-CaMBD2-b complex adopts a rigid α-helical conformation in its linker region (Figure 1). Such an extended conformation of CaM has been reported in other CaM complexes (Larsson et al, 2001; Rodriguez-Castaneda et al, 2010). …”

Section: Discussionsupporting

confidence: 80%

Structural Basis for Calmodulin as a Dynamic Calcium Sensor

et al. 2012

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Calmodulin is a prototypical and versatile Ca2+ sensor with EF-hands as its high-affinity Ca2+ binding domains. Calmodulin is present in all eukaryotic cells, mediating Ca2+-dependent signaling. Upon binding Ca2+, calmodulin changes its conformation to form complexes with a diverse array of target proteins. Despite a wealth of knowledge on calmodulin, little is known on how target proteins regulate calmodulin’s ability to bind Ca2+. Here, we take advantage of two splice variants of SK2 channels, which are activated by Ca2+-bound calmodulin, but show different sensitivity to Ca2+ for their activation. Protein crystal structures and other experiments show that depending on which SK2 splice variant it binds to calmodulin adopts drastically different conformations with different affinities for Ca2+ at its C-lobe. Such target protein induced conformational changes make calmodulin a dynamic Ca2+ sensor, capable of responding to different Ca2+ concentrations in cellular Ca2+ signaling.

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Section: Discussionsupporting

confidence: 80%

Structural Basis for Calmodulin as a Dynamic Calcium Sensor

et al. 2012

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“…It is possible that calmodulin binds Nhp6Ap in manner distinct from the standard wraparound mode, because it seems unlikely that all cytosolic proteins that interact with calmodulin are imported into the nucleus. Indeed, recent evidence suggests that calmodulin can interact with some transcription factors in an unusual dimer conformation (23). Once bound, the complex of calmodulin and Nhp6Ap probably recruits additional proteins needed to facilitate its movement across the nuclear pore.…”

Section: Discussionmentioning

confidence: 99%

“…In addition, the family of transcription factors using this pathway may extend to the helix-loop-helix family of transcription factors that also bind to calmodulin (23,26,27). The entry of nuclear proteins by calmodulin, although functionally redundant with the canonic Ran-dependent pathway, is subject to independent regulation by intracellular Ca 2ϩ mobilization.…”

Section: Discussionmentioning

confidence: 99%

The High Mobility Group Box Transcription Factor Nhp6Ap Enters the Nucleus by a Calmodulin-dependent, Ran-independent Pathway

Hanover

Love

DeAngelis

et al. 2007

Journal of Biological Chemistry

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A gradient of Ran⅐GTP typically regulates traffic through the nuclear pore by modulating association of receptors with cargo.However, here we demonstrate that the yeast high mobility group box transcription factor Nhp6Ap enters the nucleus via a novel nuclear localization signal recognized by calcium calmodulin in a process that does not require Ran. Calmodulin is strictly required for the nondiffusional nuclear entry of Nhp6Ap. Calmodulin and DNA exhibit mutually exclusive binding to NHP6A, indicating that the directionality of Nhp6Ap nuclear accumulation may be driven by DNA-dependent dissociation of calmodulin. Our findings demonstrate that calmodulin can serve as a molecular switch triggering nuclear entry with subsequent dissociation of calmodulin binding upon interaction of cargo with chromatin. This pathway appears to be evolutionarily conserved; mammalian high mobility group box transcription factors often have two nuclear localization signals: one a classical Ran-dependent signal and a second that binds calmodulin. The finding that Nhp6Ap nuclear entry requires calmodulin but not Ran indicates that Nhp6Ap is a good model for studying this poorly understood but evolutionarily conserved calmodulin-dependent nuclear import pathway.Proteins enter the nucleus through nuclear pore complexes (NPCs).2 Those smaller than ϳ40 -50 kDa can traverse these pores by passive diffusion (1-5). Many larger proteins require soluble receptors, termed importins or karyopherins, which bind cargo in the cytoplasm, accompany it through the NPC, and release it in the nucleus. Most cargo proteins contain short nuclear localization signals (NLS), motifs recognized by the various receptor proteins.The small GTPase Ran drives the accumulation of cargo proteins in the nucleus by regulating the association and dissociation of NLS-containing proteins with importin/karyopherins.In the nucleus, most Ran is GTP-bound, whereas it is largely GDP-bound in the cytoplasm. Cargo proteins bind importins in the presence of Ran-GDP in the cytoplasm, and when an importin bound to cargo arrives in the nucleus, Ran-GTP induces cargo release (6). Thus, the Ran⅐GTP gradient across the NPC drives the accumulation of most NLS-containing cargo in the nucleus.Some Ran-independent nuclear import pathways have been described, although most remain poorly understood (for review see Ref. 7). One of the most intriguing of these involves the movement of calmodulin across the nuclear pore (8 -10). Calmodulin nuclear import was argued to be a facilitated mechanism that was blocked by the calmodulin antagonist peptide M13 and did not require cytosolic factors or an ATP-regenerating system (8, 10). In the presence of calcium, calmodulin was also shown to be able to facilitate movement of large molecules into the nucleus. This latter property suggested a Ca 2ϩ -inducible nuclear import function for calmodulin that might operate during cellular activation. The calmodulin-mediated pathway was proposed to be redundant with the canonic Ran⅐GTP-driven nuclear entry pathway...

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“…Calmodulin binding to the basic sequence of the bHLH transcription factors E12, E47, and SEF2-1 and how this inhibits their DNA binding in vitro has been extensively characterized (6,7,15,19). Furthermore, increasing intracellular Ca 2ϩ by ionomycin treatment inhibits the transcriptional activity of E47 and SEF2-1 (6).…”

Section: Discussionmentioning

confidence: 99%

Calcium/Calmodulin Inhibition of Transcriptional Activity of E-proteins by Prevention of Their Binding to DNA

Saarikettu

Sveshnikova

Grundström

2004

Journal of Biological Chemistry

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The Ca 2؉ sensor protein calmodulin can interact with the DNA binding basic helix-loop-helix (bHLH) domain of E12, E47, and SEF2-1 (E2-2), which belong to the Eprotein subclass of bHLH transcription factors. This interaction inhibits the DNA binding of these bHLH proteins in vitro, and an ionophore that increases intracellular Ca 2؉ can inhibit transcriptional activation by the E-proteins. Here we have attempted to determine if these phenomena reflect a direct calmodulin-dependent inhibition of DNA binding by E-proteins in vivo. We show that calmodulin overexpression inhibits the transcriptional activity of E12, E47, and SEF2-1. We have compared calmodulin effects on DNA binding in vitro and on activation of transcription in vivo using a series of E12 mutants harboring defined alterations within the basic sequence of the bHLH domain that reduce their ability to bind calmodulin to varying degrees. We find a striking direct correlation between the ability of calmodulin to inhibit their DNA binding in vitro and the ability of overexpressed calmodulin or cellular Ca 2؉ mobilization to inhibit their transcriptional activity in vivo. Furthermore, E12 and overexpressed calmodulin were co-localized in the nucleus, and calmodulin pulldown experiments with cell extracts showed a Ca 2؉ -dependent interaction between calmodulin and E12 but not with a calmodulin inhibition-deficient E12 mutant. Chromatin immunoprecipitation showed that calmodulin overexpression leads to decreased binding of E12 and E47, but not a calmodulin inhibition-deficient E12 mutant, to the DNA recognition sequence in vivo. The data suggest that Ca 2؉ signaling can inhibit the transcriptional activities of E-proteins through direct binding of Ca 2؉ /calmodulin to the basic sequence of E-proteins, resulting in inhibition of their DNA binding.Calmodulin is a ubiquitously expressed regulator of numerous cellular processes including cell cycle progression, cell mobility and contraction, ion homeostasis, and transcription. Most target proteins bind to the Ca 2ϩ -loaded form of calmodulin. Therefore, upon signals leading to increased intracellular Ca 2ϩ concentration, calmodulin is able to bind to a new set of target proteins and modulate their activity (1). Several transcriptional regulators are direct targets for calmodulin or are regulated by calmodulin-dependent kinases or the calmodulin-dependent phosphatase calcineurin (2, 3).Basic helix-loop-helix (bHLH) 1 proteins are a large family of transcription factors that are important regulators of many cellular functions including differentiation processes such as myogenesis, hematopoiesis, and neurogenesis (4). bHLH transcription factors can be divided into several classes based on their structure, dimerization properties, function, and expression pattern. E-proteins (class I bHLH transcription factors) are encoded by three mammalian genes, E2A, SEF2-1 (also denoted E2-2), and HEB. The E2A gene encodes the alternatively spliced protein products E12 and E47 (4). E-proteins usually function as heterodimers wi...

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A novel target recognition revealed by calmodulin in complex with the basic helix–loop–helix transcription factor SEF2‐1/E2‐2

Cited by 21 publications

References 55 publications

Structural Basis for Calmodulin as a Dynamic Calcium Sensor

Structural Basis for Calmodulin as a Dynamic Calcium Sensor

The High Mobility Group Box Transcription Factor Nhp6Ap Enters the Nucleus by a Calmodulin-dependent, Ran-independent Pathway

Calcium/Calmodulin Inhibition of Transcriptional Activity of E-proteins by Prevention of Their Binding to DNA

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