Basic Helix-Loop-Helix Protein Sequences Determining Differential Inhibition by Calmodulin and S-100 Proteins

Abstract

Basic helix-loop-helix (bHLH) 1 proteins are a class of transcription factors that are important regulators in numerous systems, often involving the control of cell growth and differentiation (1). Most bHLH proteins (with some exceptions) can be broadly classified into two groups based on their patterns of expression. Class A bHLH transcription factors, also called E proteins, are broadly expressed and include the E2A gene products E12/E47 (2) and products of the E2-2/SEF2-1 gene (3, 4). E proteins are capable of forming both homodimers with themselves and heterodimers with cell type-specific class B proteins. This large group of tissue-restricted proteins includes the myogenic proteins (myogenin (5, 6), MyoD (7), MRF4 (8 -10), and Myf5 (11)) and proteins involved in neurogenesis, including MASH2 (12) and NeuroD (13). Functional activity of a class B protein in vivo requires heterodimerization with an E protein, resulting in the commitment of cells to differentiation pathways (14).Calcium plays a crucial role in many cellular processes (15, 16). Its actions are largely mediated through a family of calcium-binding proteins, of which calmodulin is the major calcium sensor. Calmodulin is a highly conserved, ubiquitously expressed protein that is essential for cell growth (17, 18). Calmodulin has four high affinity calcium binding sites called EF-hands, each composed of two ␣-helices connected by a calcium binding loop (19). Upon calcium binding, calmodulin undergoes a conformational change to expose hydrophobic patches (Ref. 20 and references therein), which allows interaction with numerous target proteins and the subsequent activation of signaling pathways.S-100 proteins are other members of the EF-hand protein family that also modulate the activities of various proteins. At least 17 members of the S-100 family have been identified. They vary in their target specificity, cell type distribution, and cell cycle regulation (21). The best studied members of the S-100 family, S-100␣ and S-100␤, have been shown to exist as both homodimers with themselves and heterodimers with each other. Their expression patterns differ; S-100␣ is predominantly found in muscle, whereas S-100␤ is highly expressed in cells within the nervous system (22-26). S-100 proteins are believed to interact with many proteins, and the identification of several common targets with calmodulin suggests that a common structural domain mediates these interactions (27)(28)(29)(30).We have previously shown that calcium-loaded calmodulin can selectively inhibit the DNA binding of E protein homodimers in vitro and that the calcium ionophore ionomycin inhibits their activity in vivo. In contrast, the heterodimers E12/MASH2 and E12/MyoD were either less sensitive or completely resistant (31).Here we identify the protein sequences within the bHLH proteins that determine the differential inhibition by calmodulin. We show that this inhibition is the result of a physical interaction between the DNA binding basic sequence and calmodulin. Both E12 and MyoD basic seq...

B-cell receptor activation inhibits AID expression through calmodulin inhibition of E-proteins

“…24, and electrophoretic mobility shift assays were performed as described in ref. 23, using an end-labeled DNA probe (5Ј-GGGAGCACAGCTGTCTCAGC-3Ј) containing the E-protein binding sequence from the AID enhancer (16,18). Western blot was performed by using the WesternBreeze immunodetection system (Invitrogen) according to the manufacturer's instructions, using antibodies to E2A (V-18), Id1 (Z-8), Id2 (C-20), Id3 (H-70), or Id4 (H-70) (all from Santa Cruz Biotechnology) or ␣-tubulin (clone B-5-1-2; Sigma-Aldrich).…”

“…We have shown, however, that Ca 2ϩ -loaded calmodulin can inhibit DNA binding of E2A and other E-proteins by directly binding to the DNA-binding basic sequences in their basic helix-loop-helix domains (22)(23)(24). Thus, we examined the possible participation of E2A in the inhibition of AID gene expression.…”

Calmodulin Regulates the Transcriptional Activity of Estrogen Receptors

“…For example, Ca 2ϩ /calmodulin-dependent kinases regulate gene transcription by altering coactivator function (26). Furthermore, calmodulin binds to members of the basic helix-loop-helix transcription factors, modifying their DNA binding (27). Recently, a family of calmodulin-binding transcription activators was identified (28).…”

Binding of Ca2+ and Zn2+ to Human Nuclear S100A2 and Mutant Proteins

“…Indeed, some S100 members activate nuclear protein kinases (e.g. S100B activates the new Ndr kinase 2 and binds to bHLH transcription factors (46,47). The question arises as to whether S100A2 has the correct Ca 2ϩ sensitivity and protein-interactive properties.…”