Memory consolidation in the cerebellar cortex

Several forms of learning, including classical conditioning of the eyeblink, depend upon the cerebellum. In examining mechanisms of eyeblink conditioning in rabbits, reversible inactivations of the control circuitry have begun to dissociate aspects of cerebellar cortical and nuclear function in memory consolidation. It was previously shown that post-training cerebellar cortical, but not nuclear, inactivations with the GABA(A) agonist muscimol prevented consolidation but these findings left open the question as to how final memory storage was partitioned across cortical and nuclear levels. Memory consolidation might be essentially cortical and directly disturbed by actions of the muscimol, or it might be nuclear, and sensitive to the raised excitability of the nuclear neurons following the loss of cortical inhibition. To resolve this question, we simultaneously inactivated cerebellar cortical lobule HVI and the anterior interpositus nucleus of rabbits during the post-training period, so protecting the nuclei from disinhibitory effects of cortical inactivation. Consolidation was impaired by these simultaneous inactivations. Because direct application of muscimol to the nuclei alone has no impact upon consolidation, we can conclude that post-training, consolidation processes and memory storage for eyeblink conditioning have critical cerebellar cortical components. The findings are consistent with a recent model that suggests the distribution of learning-related plasticity across cortical and nuclear levels is task-dependent. There can be transfer to nuclear or brainstem levels for control of high-frequency responses but learning with lower frequency response components, such as in eyeblink conditioning, remains mainly dependent upon cortical memory storage

The Transcription Factor YY1 Is a Substrate for Polo-Like Kinase 1 at the G2/M Transition of the Cell Cycle

Yin-Yang 1 (YY1) is an essential multifunctional zinc-finger protein. It has been shown over the past two decades to be a critical regulator of a vast array of biological processes, including development, cell proliferation and differentiation, DNA repair, and apoptosis. YY1 exerts its functions primarily as a transcription factor that can activate or repress gene expression, dependent on its spatial and temporal context. YY1 regulates a large number of genes involved in cell cycle transitions, many of which are oncogenes and tumor-suppressor genes. YY1 itself has been classified as an oncogene and was found to be upregulated in many cancer types. Unfortunately, our knowledge of what regulates YY1 is very minimal. Although YY1 has been shown to be a phosphoprotein, no kinase has ever been identified for the phosphorylation of YY1. Polo-like kinase 1 (Plk1) has emerged in the past few years as a major cell cycle regulator, particularly for cell division. Plk1 has been shown to play important roles in the G/M transition into mitosis and for the proper execution of cytokinesis, processes that YY1 has been shown to regulate also. Here, we present evidence that Plk1 directly phosphorylates YY1 in vitro and in vivo at threonine 39 in the activation domain. We show that this phosphorylation is cell cycle regulated and peaks at G2/M. This is the first report identifying a kinase for which YY1 is a substrate

Threonine 39 phosphorylation on YY1 peaks at G2/M transition.

<p>Stable HeLa-Flag-YY1 cells were synchronized by double-thymidine block and then released. (<b>A</b>) Analysis of the cell-cycle progression of HeLa cells released after double-thymidine block using fluorescence-activated cell sorting. Cells were stained with propidium iodide and analyzed based on their DNA content. An asynchronous population of cells was used as a control. (<b>B</b>) Whole cell extracts were prepared from HeLa-Flag-YY1 cells collected at the indicated times after release from double-thymidine block. Total WCE were analyzed on a Western blot after SDS-PAGE separation, and probed with anti-Plk1, anti-Cyclin B1, and anti-YY1 antibodies. Flag-YY1 was immunoprecipitated from the extracts of each time point, and then analyzed on a Western blot using anti-pT39 and anti-YY1 antibodies.</p

Plk1 phosphorylates YY1 in the N-terminal activation domain <i>in vitro</i>.

<p>(<b>A</b>) Coomassie blue staining (left) and phosphorimager exposure (right) of SDS-PAGE gel analysis of the radioactive <i>in vitro</i> kinase assay reactions using purified Plk1 and a panel of GST-tagged YY1 deletion mutants. The specific YY1 deletions are indicated above the lanes. Equal amounts of purified Plk1 were added to all reactions. (<b>B</b>) Diagram of the deletion mutants of YY1 used in the kinase assay in (A). Evidence of phosphorylation shown in (A) is indicated by the (+) sign, whereas the absence of evidence of phosphorylation is indicated by a (--) sign. The region identified as the site for phosphorylation by Plk1 is indicated (amino acid residues 2–62).</p

Plk1 phosphorylates YY1 at threonine 39 <i>in vitro</i>.

<p>(<b>A</b>) Diagram displaying the different domains of the YY1 protein. Amino acid residues 2–62 are shown; serine and threonine residues in amino acids 2–62 are indicated by arrows. The predicted phosphorylation site at threonine 39 is indicated with a star. (<b>B</b>) Coomassie blue staining and phosphorimager exposure of the SDS-PAGE gel analysis of the radioactive <i>in vitro</i> kinase assay reactions. Kinase reactions include Plk1 only (no substrate lane), Plk1 with GST-YY1 wild type (WT) or mutant (T39A). (<b>C</b>) Amino acid sequence alignment of the N-terminal domain of the YY1 protein from different species, as indicated.</p

Anti-p-T39 antibody specifically recognizes YY1 phosphorylation at threonine 39.

<p>(<b>A</b>) Western blot analysis of the cold <i>in vitro</i> kinase assay reactions using purified Plk1 and purified YY1, after SDS-PAGE. The blot was probed with anti-YY1, anti-Plk1 and anti-pT39 antibodies. (<b>B</b>) Western blot analysis of the cold <i>in vitro</i> kinase assay reactions after SDS-PAGE, using purified Plk1 and purified GST-YY1 wild type (WT) or mutant (T39A). The blot was probed with anti-YY1, anti-Plk1 and anti-pT39 antibodies.</p