In mammals, growth of the fetal heart is regulated by proliferation of cardiac muscle cells. At later stages of pre-natal life, this proliferation diminishes profoundly [1] [2] and the dramatic expansion in heart size during the transition to adulthood is due exclusively to hypertrophy of individual cardiomyocytes [3] [4] [5]. Cardiomyocyte hypertrophy also contributes to the pathology of most post-natal heart disease [6] [7] [8] [9] [10]. Within this context, numerous signal transduction pathways have been implicated as the link between the effector(s) and altered cardiac gene expression [11] [12] [13] [14] [15] [16]. A common pathway has yet to be discovered, however. Here, we found that the activity of the stress-activated kinase p38 was enhanced in both types of cardiomyocyte hypertrophy. We also found that a target of the activated p38 kinase is the cardiac transcription factor MEF2. Transgenic mice expressing a dominant-negative form of MEF2C displayed attenuated post-natal growth of the myocardium. These results provide the first evidence for a single pathway regulating both normal and pathologic cardiomyocyte hypertrophy.

Spinal muscular atrophy (SMA) is characterized by motor neuron loss, caused by mutations or deletions in the ubiquitously expressed survival motor neuron 1 (SMN1) gene. We recently identified a novel role for Smn protein in glucose metabolism and pancreatic development in both an intermediate SMA mouse model (Smn2B/−) and type I SMA patients. In the present study, we sought to determine if the observed metabolic and pancreatic defects are SMA-dependent. We employed a line of heterozygous Smn-depleted mice (Smn+/−) that lack the hallmark SMA neuromuscular pathology and overt phenotype. At 1 month of age, pancreatic/metabolic function of Smn+/−mice is indistinguishable from wild type. However, when metabolically challenged with a high-fat diet, Smn+/−mice display abnormal localization of glucagon-producing α-cells within the pancreatic islets and increased hepatic insulin and glucagon sensitivity, through increased p-AKT and p-CREB, respectively. Further, aging results in weight gain, an increased number of insulin-producing β cells, hyperinsulinemia and increased hepatic glucagon sensitivity in Smn+/−mice. Our study uncovers and highlights an important function of Smn protein in pancreatic islet development and glucose metabolism, independent of canonical SMA pathology. These findings suggest that carriers of SMN1 mutations and/or deletions may be at an increased risk of developing pancreatic and glucose metabolism defects, as even small depletions in Smn protein may be a risk factor for diet- and age-dependent development of metabolic disorders.

The beta‐globin locus control region (LCR) confers high levels of position‐independent, copy number‐dependent expression onto globin transgenes. Here > 40 independent transgenic mouse lines and founders that carried the LCR in cis with the beta‐globin gene promoter driving a lacZ reporter gene were studied. Expression of the lacZ transgene was assayed by measuring beta‐galactosidase enzyme activity in fetal liver extracts, the levels of which correlated with the quantity of lacZ mRNA determined using RNase protection assays. Unexpectedly, expression of the lacZ transgene was found to show strong position effects, varying as much as 700‐fold per transgene copy. These position effects occurred even if the whole beta‐globin gene was incorporated as part of the lacZ reporter gene. Moreover, DNase I‐hypersensitive sites appeared in the transgene LCR in high expressing but not in low expressing lines, suggesting that the LCR itself was position dependent. In contrast, MEL cell clones, in which transcriptionally active integration sites were selected for, gave < 13‐fold variation in expression per copy of an LCR‐lacZ construct. These results show that the lacZ reporter affects the ability of the LCR to activate chromatin in mice and that culture cells are not an adequate model for position‐independent gene expression studies.

Recent advances in the characterization of the phosphoproteome have been limited to measuring phosphorylation statuses, which imply but do not measure protein kinase activity directly. As such, the ability to screen, compare, and define multiple protein enzymatic activities across divergent samples remains a daunting challenge in proteomics. Here, we describe a gel-based kinase assay coupled to MS identification as an approach to map global kinase activity and assign pathway architecture to specified biologic contexts. We demonstrate the utility of this method as a platform for the comparison of proteomes based on differences in both kinase activities and for use in the de novo substrate identification for individual kinases. This approach allowed us to map the signal perturbations in the post-natal heart that were associated with activation of a myopathic cascade as mediated by the mitogen-activated protein kinase MKK6 and established the novel observation that MKK6 promotes the development of cardiomyopathy through multiple substrate interactions. Serial modification of proteins is a common mechanism for eukaryotic cellular adaptation. Within this context, protein phosphorylation is believed to be the most common form of modification. Many excellent methods describing the purification and identification of phospho-proteins are now available and have significantly contributed to our understanding of cellular regulation through signal transduction (1-4). Nevertheless, phosphorylation status alone does not unambiguously characterize the kinetic steps of signaling cascades, namely the activity of protein kinases. Indeed, there are an estimated 519 kinase-related gene products in the human genome (representing ϳ3% of the coded proteome) with a unique substrate range for each kinase (5). As such, successfully mapping these steps in any biologic model remains a daunting challenge for proteomics.In addition to the limitation imposed by current screening methodologies, the accurate reconstruction of signaling networks may also be hindered by the prevailing hypothesis that describes signaling cascades as insulated and linear events. For example, the annotation of mitogen-activated protein kinase (MAPK) 1 pathways describes a three-tier modular structure involving sequential activation from module to module (6). The p38 MAPK pathway is a typical representation of this kinase family. This pathway contains a phosphorylation sequence initiated by a MAPK kinase kinase (MKKK), which activates a MAPK kinase (MKK), which then in turn activates a MAPK (p38), with the MAPK then serving as the effector enzyme to stimulate or repress the activity of corresponding protein substrates by targeted phosphorylation (7).Considerable experimental evidence supports the basic premise of the linear MKK/p38 MAPK pathway, yet ascribing all phenotypic outcomes exclusively to the activity of the effector kinase is most likely an oversimplification. In this regard, the relationship between activation of p38 signal cascades and the development of c...

Recent evidence suggests that cell cycle proteins may have novel functions beyond the control of cell division. We have investigated the role of Rb/E2F pathway in the regulation of neuronal differentiation and migration during late embryonic development.We show that loss of Rb leads to terminal differentiation and radial migration defects as well as loss of specific interneuron subtypes in the developing forebrain and olfactory bulb. This phenotype is linked to a dramatic reduction in the levels of Dlx homeodomain genes that regulate ventral telencephalic development, most significantly Dlx2. To ask if Rb plays a direct role in controlling the induction of Dlx2, we examined the regulatory regions of the Dlx1/Dlx2 locus. Using chromatin immunoprecipitation experiments, we show that Rb modulates Dlx gene expression through interaction with the Dlx forebrain-specific enhancer, I12b, the Dlx2 proximal promoter and 3'UTR region in vivo. This interaction is mediated by E2F functional sites located in I12b that act as repressor sites. Deletion of E2F consensus sites on the I12b-Dlx1/Dlx2 enhancer results in increased reporter activity in the subventricular zone of the developing brain. We demonstrate that in the absence of Rb, E2F7, an Rb-independent repressor, is upregulated in the brain and could ectopically repress the I12b activity and Dlx2 transcription.In conclusion, our data provides the first evidence that cell cycle proteins such as Rb play an essential role to coordinate the transition from proliferation to differentiation and maintain terminal differentiation by regulating the levels of key transcription factors such as Dlx2 during neurogenesis. This work was supported by a grant from CIHR.The specification of unique neuronal and glial sub-types relies upon the restricted expression of cell fate determinants, acting primarily in 'combinatorial codes'. The expression of the proper combinatorial code in each sub-type in turn relies upon the combinatorial action of upstream regulators, acting in a positional and temporal manner. While many players acting at these different levels have been identified, the complex multi-step regulatory flow acting to dictate any one unique cell fate in the nervous system has not been deciphered. Moreover, because each sub-type is generated in distinct numbers, such in-depth decoding of the neural diversification process also requires addressing how upstream and downstream combinatorial codes intersect with the cell cycle and cell death machineries.To address these issues, we are using a specific Drosophila CNS progenitor cell, the neuroblast 5-6, as a model. This neuroblast is presented in all 18 segments of the developing fly CNS, but generates a unique group of neurons, the Apterous cluster of four neurons, only in the three thoracic segments. Two of the Ap cluster cells are furthermore uniquely identifiable by their selective expression of the FMRFa and Nplp1 neuropeptide genes. Specification of Ap neurons requires a complex interplay between positional and temporal cues, whic...