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Docking to a Basic Helix Promotes Specific Phosphorylation by G1-Cdk1.

ABSTRACT: Cyclins are the activators of cyclin-dependent kinase (CDK) complex, but they also act as docking scaffolds for different short linear motifs (SLiMs) in CDK substrates and inhibitors. According to the unified model of CDK function, the cell cycle is coordinated by CDK both via general CDK activity thresholds and cyclin-specific substrate docking. Recently, it was found that the G1-cyclins of S. cerevisiae have a specific function in promoting polarization and growth of the buds, making the G1 cyclins essential for cell survival. Thus, while a uniform CDK specificity of a single cyclin can be sufficient to drive the cell cycle in some cells, such as in fission yeast, cyclin specificity can be essential in other organisms. However, the known G1-CDK specific LP docking motif, was not responsible for this essential function, indicating that G1-CDKs use yet other unknown docking mechanisms. Here we report a discovery of a G1 cyclin-specific (Cln1,2) lysine-arginine-rich helical docking motif (the K/R motif) in G1-CDK targets involved in the mating pathway (Ste7), transcription (Xbp1), bud morphogenesis (Bud2) and spindle pole body (Spc29, Spc42, Spc110, Sli15) function of S. cerevisiae. We also show that the docking efficiency of K/R motif can be regulated by basophilic kinases such as protein kinase A. Our results further widen the list of cyclin specificity mechanisms and may explain the recently demonstrated unique essential function of G1 cyclins in budding yeast.

SUBMITTER: Faustova I

PROVIDER: S-EPMC8431026 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Cyclin-specific docking motifs promote phosphorylation of yeast signaling proteins by G1/S Cdk complexes.

Project description:The eukaryotic cell cycle begins with a burst of cyclin-dependent kinase (Cdk) phosphorylation. In budding yeast, several Cdk substrates are preferentially phosphorylated at the G1/S transition rather than later in the cell cycle when Cdk activity levels are high. These early Cdk substrates include signaling proteins in the pheromone response pathway. Two such proteins, Ste5 and Ste20, are phosphorylated only when Cdk is associated with the G1/S cyclins Cln1 and Cln2 and not G1, S, or M cyclins. The basis of this cyclin specificity is unknown.Here we show that Ste5 and Ste20 have recognition sequences, or "docking" sites, for the G1/S cyclins. These docking sites, which are distinct from Clb5/cyclin A-binding "RXL" motifs, bind preferentially to Cln2. They strongly enhance Cln2-driven phosphorylation of each substrate in vivo and function largely independent of position and distance to the Cdk sites. We exploited this functional independence to rewire a Cdk regulatory circuit in a way that changes the target of Cdk inhibition in the pheromone response pathway. Furthermore, we uncover functionally active Cln2 docking motifs in several other Cdk substrates. The docking motifs drive cyclin-specific phosphorylation, and the cyclin preference can be switched by using a distinct motif.Our findings indicate that some Cdk substrates are intrinsically capable of being phosphorylated by several different cyclin-Cdk forms, but they are inefficiently phosphorylated in vivo without a cyclin-specific docking site. Docking interactions may play a prevalent but previously unappreciated role in driving phosphorylation of select Cdk substrates preferentially at the G1/S transition.

| S-EPMC3196376 | biostudies-literature

Phosphorylation of the Twist1-family basic helix-loop-helix transcription factors is involved in pathological cardiac remodeling.

Project description:BackgroundThe Twist1-family basic helix-loop-helix (bHLH) transcription factors including Twist1, Hand1 and Hand2, play an essential role in heart development and are implicated in pathological heart remodeling. Previously, it was reported that these bHLH transcription factors can be regulated by phosphorylation within the basic-helix I domain, which is involved in developmental processes such as limb formation and trophoblast differentiation. However, how phosphorylation of Twist1 family functions in post-natal heart is elusive.Principal findingsHere, we generated transgenic mice with over-expression of Hand1 and Twist1 mutants (to mimic or to abolish phosphorylation) in cardiomyocytes and found pathological cardiac remodeling leading to heart failure and sudden death. Gene expression profile analysis revealed up-regulation of growth-promoting genes and down-regulation of metabolic genes. It is well known that aberrant activation of Akt signaling causes pathological cardiac remodeling and results in heart failure. The basic-helix I domain of Twist1 family members contain Akt substrate consensus motif and may be downstream targets of Akt signaling. Using biochemical analysis, we demonstrated that Hand1 and Twist1 were phosphorylated by Akt in the basic-helix I domain. Phosphorylation of Hand1 regulated its transcriptional activation of luciferase reporter genes and DNA binding ability.ConclusionsThis study provides novel insights into the regulation of Twist1 family in cardiac remodeling and suggests that the Twist1 family can be regulated by Akt signaling.

| S-EPMC3084786 | biostudies-literature

The grape berry-specific basic helix-loop-helix transcription factor VvCEB1 affects cell size.

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Myc phosphorylation in its basic helix-loop-helix region destabilizes transient ?-helical structures, disrupting Max and DNA binding.

Project description:Myelocytomatosis proto-oncogene transcription factor (Myc) is an intrinsically disordered protein with critical roles in cellular homeostasis and neoplastic transformation. It is tightly regulated in the cell, with Myc phosphorylation playing a major role. In addition to the well-described tandem phosphorylation of Thr-52 and Ser-62 in the Myc transactivation domain linked to its degradation, P21 (RAC1)-activated kinase 2 (PAK2)-mediated phosphorylation of serine and threonine residues in the C-terminal basic helix-loop-helix leucine zipper (bHLH-LZ) region regulates Myc transcriptional activity. Here we report that PAK2 preferentially phosphorylates Myc twice, at Thr-358 and Ser-373, with only a minor fraction being modified at the previously identified Thr-400 site. For transcriptional activity, Myc binds E-box DNA elements, requiring its heterodimerization with Myc-associated factor X (Max) via the bHLH-LZ regions. Using isothermal calorimetry (ITC), we found that Myc phosphorylation destabilizes this ternary protein-DNA complex by decreasing Myc's affinity for Max by 2 orders of magnitude, suggesting a major effect of phosphorylation on this complex. Phosphomimetic substitutions revealed that Ser-373 dominates the effect on Myc-Max heterodimerization. Moreover, a T400D substitution disrupted Myc's affinity for Max. ITC, NMR, and CD analyses of several Myc variants suggested that the effect of phosphorylation on the Myc-Max interaction is caused by secondary structure disruption during heterodimerization rather than by a change in the structurally disordered state of Myc or by phosphorylation-induced electrostatic repulsion in the heterodimer. Our findings provide critical insights into the effects of PAK2-catalyzed phosphorylation of Myc on its interactions with Max and DNA.

| S-EPMC6005445 | biostudies-literature

A basic helix-loop-helix transcription factors network mediates the brassinosteroid signal

Project description:This SuperSeries is composed of the SubSeries listed below.

2014-03-05 | GSE51121 | GEO

Prg1 is regulated by the basic helix-loop-helix transcription factor Math2

Project description:Math2 (NEX-1/NeuroD6) is a member of the bHLH transcription factor family and is involved in neuronal differentiation and maturation. In the present study, we identified the genes targeted by Math2 using DNA microarrays and cultured rat cortical cells transfected with Math2. Of the genes regulated by Math2, we focused on plasticity-related gene 1 (Prg1). Prg1 expression induced by Math2 was confirmed in cultured rat cortical cells and PC12 cells analyzed by real-time quantitative PCR. Examining the promoter region of rat Prg1, we found four E-boxes designated -E1 to -E4 (CANNTG) which were recognized by the bHLH transcription factor. Using chromatin immunoprecipitation (ChIP) assays, we found that Math2 directly bound to the E-box(es) in the Prg1 promoter. The reporter assay of Prg1 showed that -E1 was critical for the regulation of the Prg1 expression by Math2. Then, the functional role of Math2 and Prg1 was investigated in PC12 cells. Seventy-two hours after transfection of Math2 or Prg1, neurite length and number was significantly induced in PC12 cells. Co-transfection with Prg1-siRNA completely inhibited Math2-mediated morphological changes. Our results suggest that Math2 directly regulates Prg1 expression and Math2-Prg1 cascade plays an important role in neurite outgrowth in PC12 cells.

2008-07-27 | GSE11829 | GEO

Isoform-Specific Reduction of the Basic Helix-Loop-Helix Transcription Factor TCF4 Levels in Huntington's Disease.

Project description:Huntington's disease (HD) is an inherited neurodegenerative disorder with onset of characteristic motor symptoms at midlife, preceded by subtle cognitive and behavioral disturbances. Transcriptional dysregulation emerges early in the disease course and is considered central to HD pathogenesis. Using wild-type (wt) and HD knock-in mouse striatal cell lines we observed a HD genotype-dependent reduction in the protein levels of transcription factor 4 (TCF4), a member of the basic helix-loop-helix (bHLH) family with critical roles in brain development and function. We characterized mouse Tcf4 gene structure and expression of alternative mRNAs and protein isoforms in cell-based models of HD, and in four different brain regions of male transgenic HD mice (R6/1) from young to mature adulthood. The largest decrease in the levels of TCF4 at mRNA and specific protein isoforms were detected in the R6/1 mouse hippocampus. Translating this finding to human disease, we found reduced expression of long TCF4 isoforms in the postmortem hippocampal CA1 area and in the cerebral cortex of HD patients. Additionally, TCF4 protein isoforms showed differential synergism with the proneural transcription factor ASCL1 in activating reporter gene transcription in hippocampal and cortical cultured neurons. Induction of neuronal activity increased these synergistic effects in hippocampal but not in cortical neurons, suggesting brain region-dependent differences in TCF4 functions. Collectively, this study demonstrates isoform-specific changes in TCF4 expression in HD that could contribute to the progressive impairment of transcriptional regulation and neuronal function in this disease.

| S-EPMC8519306 | biostudies-literature

SclR, a basic helix-loop-helix transcription factor, regulates hyphal morphology and promotes sclerotial formation in Aspergillus oryzae.

Project description:Most known basic-region helix-loop-helix (bHLH) proteins belong to a superfamily of transcription factors often involved in the control of growth and differentiation. Therefore, inappropriate expression of genes encoding bHLH proteins is frequently associated with developmental dysfunction. In our previously reported study, a novel bHLH protein-encoding gene (AO090011000215) of Aspergillus oryzae was identified. The gene-disrupted strain was found to produce dense conidia, but sparse sclerotia, relative to the parent strain. Here, to further analyze its function, we generated an overexpressing strain using the A. oryzae amyB gene promoter. Genetic overexpression led to a large number of initial hyphal aggregations and then the formation of mature sclerotia; it was therefore designated sclR (sclerotium regulator). At the same time, the sclR-overexpressing strain also displayed both delayed and decreased conidiation. Scanning electron microscopy indicated that the aerial hyphae of the sclR-overexpressing strain were extremely branched and intertwined with each other. In the generation of the SclR-enhanced green fluorescent protein (EGFP) expression strain, the SclR-EGFP protein fusion was conditionally detected in the nuclei. In addition, the loss of sclR function led to rapid protein degradation and cell lysis in dextrin-polypeptone-yeast extract liquid medium. Taken together, these observations indicate that SclR plays an important role in hyphal morphology, asexual conidiospore formation, and the promotion of sclerotial production, even retaining normal cell function, at least in submerged liquid culture.

| S-EPMC3147411 | biostudies-literature

A basic helix-loop-helix transcription factors network mediates the brassinosteroid signal

Project description:This SuperSeries is composed of the SubSeries listed below. Refer to individual Series

2014-03-05 | E-GEOD-51121 | biostudies-arrayexpress

The basic helix-loop-helix transcription factor Mist1 overexpression in mouse fetal hepatoblast culture

Project description:To identify the molecular mechanism of Mist1 during hepatoblasts differentiation, we performed gene expression analysis in Mist1 over-expressed cells. Dlk+ hepatoblasts derived from E13 fetal livers were purified. Purification of Dlk+ cells were performed using anti-Dlk antibody after hematopoietic cells were removed. Cells were infected with mock or Mist1-overexpressing retroviruses and cultured for 3days with oncostatin M.

2015-03-31 | GSE67406 | GEO

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