We have examined the role of protein phosphorylation in the modulation of the key muscle-specific transcription factor MyoD. We show that MyoD is highly phosphorylated in growing myoblasts and undergoes substantial dephosphorylation during differentiation. MyoD can be efficiently phosphorylated in vitro by either purified cdk1-cyclin B or cdk1 and cdk2 immunoprecipitated from proliferative myoblasts. Comparative two-dimensional tryptic phosphopeptide mapping combined with site-directed mutagenesis revealed that cdk1 and cdk2 phosphorylate MyoD on serine 200 in proliferative myoblasts. In addition, when the seven prolinedirected sites in MyoD were individually mutated, only substitution of serine 200 to a nonphosphorylatable alanine (MyoD-Ala200) abolished the slower-migrating hyperphosphorylated form of MyoD, seen either in vitro after phosphorylation by cdk1-cyclin B or in vivo following overexpression in 10T1/2 cells. The MyoDAla200 mutant displayed activity threefold higher than that of wild-type MyoD in transactivation of an E-box-dependent reporter gene and promoted markedly enhanced myogenic conversion and fusion of 10T1/2 fibroblasts into muscle cells. In addition, the half-life of MyoD-Ala200 protein was longer than that of wild-type MyoD, substantiating a role of Ser200 phosphorylation in regulating MyoD turnover in proliferative myoblasts. Taken together, our data show that direct phosphorylation of MyoD Ser200 by cdk1 and cdk2 plays an integral role in compromising MyoD activity during myoblast proliferation.Skeletal muscle differentiation is characterized by withdrawal of myoblasts from the cell cycle, induction of musclespecific gene expression, and cell fusion into multinucleated myotubes. All of these events are coordinated by a family of muscle-specific transcription factors including MyoD (8), Myf5 (4), myogenin (12, 56), and MRF4 (39). These proteins show homology within a basic helix-loop-helix (bHLH) domain that mediates both heterodimerization with ubiquitous activating bHLH proteins such as E12 and E47 and DNA binding to a specific sequence, CANNTG, called the E box (9,25,30). One of the most remarkable properties of myogenic factors is that their ectopic expression in nonmuscle cells forces these cells into muscle differentiation, a process known as myogenic conversion (6, 8). Although capable of inhibiting cell proliferation (7, 47) and inducing differentiation, MyoD is constitutively expressed in proliferating myoblasts long before differentiation takes place, implying that its activity is regulated in replicating cells (26,49). Indeed, when cultured myoblasts are exposed to serum or growth factors such as basic fibroblast growth factor and transforming growth factor ␤, both muscle differentiation and MyoD activity are inhibited (34, 48). One of the inhibitory mechanisms that target MyoD in proliferative myoblasts involves the Id family of proteins. These HLH proteins, which are devoid of DNA-binding basic domains, can heterodimerize with bHLH factors, thus inhibiting their binding to DN...

During Drosophila oogenesis, Notch function regulates the transition from mitotic cell cycle to endocycle in follicle cells at stage 6. Loss of either Notch function or its ligand Delta (Dl) disrupts the normal transition; this disruption causes mitotic cycling to continue and leads to an overproliferation phenotype. In this context, the only known cell cycle component that responds to the Notch pathway is String/Cdc25 (Stg), a G2/M cell cycle regulator. We found that prolonged expression of string is not sufficient to keep cells efficiently in mitotic cell cycle past stage 6, suggesting that Notch also regulates other cell cycle components in the transition. By using an expression screen, we found such a component: Fizzy-related/Hec1/Cdh1 (Fzr), a WD40 repeat protein. Fzr regulates the anaphase-promoting complex/cyclosome (APC/C) and is expressed at the mitotic-to-endocycle transition in a Notch-dependent manner. Mutant clones of Fzr revealed that Fzr is dispensable for mitosis but essential for endocycles. Unlike in Notch clones, in Fzr mutant cells mitotic markers are absent past stage 6. Only a combined reduction of Fzr and ectopic Stg expression prolongs mitotic cycles in follicle cells, suggesting that these two cell cycle regulators, Fzr and Stg, are important mediators of the Notch pathway in the mitotic-to-endocycle transition.

Background Excessive pro-inflammatory activation following trauma plays a role in late morbidity and mortality including the development of multiple organ dysfunction syndrome (MODS). To date, identification of patients at risk has been challenging. Results from animal and human studies suggest that circulating IL-6, may serve as a biomarker for excessive inflammation. The purpose of this analysis was to determine the association of IL-6 to outcome in a multi-center developmental cohort and in a single-center validation cohort. Methods Severely injured patients with shock due to hemorrhage were evaluated within a multi-center developmental cohort (n=79). All had blood drawn within 12 hours of injury. Plasma IL-6 was determined by multiplex proteomic analysis. Clinical and outcome data were prospectively obtained. Within this developmental cohort, a plasma IL-6 level was determined for the subsequent development of MODS by developing a receiver operating curve (ROC) and defining the optimal IL-6 level using the Youden index. This IL-6 level was then evaluated within a separate validation cohort (n=56). Results A receiver operating curve was generated for IL-6 and MODS development with an IL-6 level of 350 pg/ml having the highest sensitivity and specificity within the developmental cohort. IL-6 was associated with MODS after adjusting for APACHE II, ISS, male gender and blood transfusions with an odds ratio of 3.9 [95% CI: 1.33–11.19]. An IL-6 level greater than 350 pg/ml within the validation cohort was associated with an increase in MODS score, MODS development, ventilator days, ICU length of stay (LOS), and hospital LOS. However, this IL-6 level was not associated with either the development of nosocomial infection or mortality. Conclusion Elevation in plasma IL-6 appears to correlate with a poor prognosis. This measurement may be useful as a biomarker for prognosis and serve to identify patients at higher risk of adverse outcome that would benefit from novel therapeutic interventions.

(LAMB2) is a critical component of the glomerular basement membrane as content of LAMB2 in part determines glomerular barrier permeability. Previously, we reported that high concentrations of glucose reduce expression of this laminin subunit at the translational level. The present studies were undertaken to further define systems that control Lamb2 translation and the effect of high glucose on those systems. Complementary studies were performed using in vitro differentiation of cultured podocytes and mesangial cells exposed to normal and elevated concentrations of glucose, and tissues from control and diabetic rats. Together, these studies provide evidence for regulation of Lamb2 translation by IMP2, an RNA binding protein that targets Lamb2 mRNA to the actin cytoskeleton. Expression of Imp2 itself is regulated by the transcription factor HMGA2, which in turn is regulated by the microRNA let-7b. Elevated concentrations of glucose increase let-7b, which reduces HMGA2 expression, in turn reducing IMP2 and LAMB2. Correlative changes in kidney tissues from control and streptozotocin-induced diabetic rats suggest these control mechanisms are operative in vivo and may contribute to proteinuria in diabetic nephropathy. To our knowledge, this is the first time that translation of Lamb2 mRNA has been linked to the actin cytoskeleton, as well as to specific RNA-binding proteins. These translational control points may provide new targets for therapy in proteinuric disorders such as diabetic nephropathy where LAMB2 levels are reduced.microRNA; diabetes LAMININS ARE HETEROTRIMERIC (␣␤␥), extracellular glycoproteins in glomerular (GBM) and other basement membranes (16). The laminin-␤2 (LAMB2) subunit is required for normal GBM structure and barrier function as human mutations causing loss of this protein lead to nephrotic syndrome and progressive kidney dysfunction (23,28,29,33). Mice deficient in LAMB2 die soon after birth with severe proteinuria and abnormal neuromuscular junctions (32, 33). Thus, LAMB2 is a key subunit in the laminin family, providing critical functions in kidney and other tissues (40).Mechanisms controlling expression of the Lamb2 gene as well as LAMB2 protein production, secretion, and degradation are not well-understood (17). We previously demonstrated that LAMB2 is not expressed in developing glomeruli of hyperglycemic animals, and it is reduced in kidneys of rats with streptozotocin-induced diabetes (2), as a result of glucosemediated impairment of Lamb2 mRNA translation (39). Also, loss of LAMB2 results in impaired migratory responses of mesangial cells (MC), which may limit repair in the face mesangiolysis, a prominent feature of diabetic nephropathy (DN) (39). Glucose-mediated reductions in LAMB2 content in GBM may also contribute to microalbuminuria in DN (30).In this report, we demonstrate that the RNA-binding protein (RNA-BP) IMP2 is required for LAMB2 translation, as 1) Lamb2 mRNA binds specifically to IMP2 and associates with actin during translation, 2) LAMB2 protein production is reduced if ...