Yong Chao Ma

Src Tyrosine Kinase Is a Novel Direct Effector of G Proteins

Heterotrimeric G proteins transduce signals from cell surface receptors to modulate the activity of cellular effectors. Src, the product of the first characterized proto-oncogene and the first identified protein tyrosine kinase, plays a critical role in the signal transduction of G protein-coupled receptors. However, the mechanism of biochemical regulation of Src by G proteins is not known. Here we demonstrate that Galphas and Galphai, but neither Galphaq, Galpha12 nor Gbetay, directly stimulate the kinase activity of downregulated c-Src. Galphas and Galphai similarly modulate Hck, another member of Src-family tyrosine kinases. Galphas and Galphai bind to the catalytic domain and change the conformation of Src, leading to increased accessibility of the active site to substrates. These data demonstrate that the Src family tyrosine kinases are direct effectors of G proteins.

Survival factor-mediated BAD phosphorylation raises the mitochondrial threshold for apoptosis.

Growth factor suppression of apoptosis correlates with the phosphorylation and inactivation of multiple proapoptotic proteins, including the BCL-2 family member BAD. However, the physiological events required for growth factors to block cell death are not well characterized. To assess the contribution of BAD inactivation to cell survival, we generated mice with point mutations in the BAD gene that abolish BAD phosphorylation at specific sites. We show that BAD phosphorylation protects cells from the deleterious effects of apoptotic stimuli and attenuates death pathway signaling by raising the threshold at which mitochondria release cytochrome c to induce cell death. These findings establish a function for endogenous BAD phosphorylation, and elucidate a mechanism by which survival kinases block apoptosis in vivo.

Hyaluronan blocks oligodendrocyte progenitor maturation and remyelination through TLR2

Failure of remyelination is largely responsible for sustained neurologic symptoms in multiple sclerosis (MS). MS lesions contain hyaluronan deposits that inhibit oligodendrocyte precursor cell (OPC) maturation. However, the mechanism behind this inhibition is unclear. We report here that Toll-like receptor 2 (TLR2) is expressed by oligodendrocytes and is up-regulated in MS lesions. Pathogen-derived TLR2 agonists, but not agonists for other TLRs, inhibit OPC maturation in vitro. Hyaluronan-mediated inhibition of OPC maturation requires TLR2 and MyD88, a TLR2 adaptor molecule. Ablated expression of TLR2 also enhances remyelination in a lysolecithin animal model. Hyaluronidases expressed by OPCs degrade hyaluronan to hyaluronan oligomers, a requirement for hyaluronan/TLR2 signaling. MS lesions contain both TLR2+ oligodendrocytes and low-molecular-weight hyaluronan, consistent with their importance to remyelination in MS. We thus have defined a mechanism controlling remyelination failure in MS where hyaluronan is degraded by hyaluronidases into hyaluronan oligomers that block OPC maturation and remyelination through TLR2-MyD88 signaling.

Regulation of Motor Neuron Specification by Phosphorylation of Neurogenin 2

The mechanisms by which proneural basic helix-loop-helix (bHLH) factors control neurogenesis have been characterized, but it is not known how they specify neuronal cell-type identity. Here, we provide evidence that two conserved serine residues on the bHLH factor neurogenin 2 (Ngn2), S231 and S234, are phosphorylated during motor neuron differentiation. In knockin mice in which S231 and S234 of Ngn2 were mutated to alanines, neurogenesis occurs normally, but motor neuron specification is impaired. The phosphorylation of Ngn2 at S231 and S234 facilitates the interaction of Ngn2 with LIM homeodomain transcription factors to specify motor neuron identity. The phosphorylation-dependent cooperativity between Ngn2 and homeodomain transcription factors may be a general mechanism by which the activities of bHLH and homeodomain proteins are temporally and spatially integrated to generate the wide diversity of cell types that are a hallmark of the nervous system.

Csk, a critical link of g protein signals to actin cytoskeletal reorganization.

Heterotrimeric G proteins can signal to reorganize the actin cytoskeleton, but the mechanism is unclear. Here we report that, in tyrosine kinase Csk-deficient mouse embryonic fibroblast cells, G protein (Gbetagamma, Galpha(12), Galpha(13), and Galpha(q))-induced, and G protein-coupled receptor-induced, actin stress fiber formation was completely blocked. Reintroduction of Csk into Csk-deficent cells restored the G protein-induced actin stress fiber formation. Chemical rescue experiments with catalytic mutants of Csk demonstrated that the catalytic activity of Csk was required for this process. Furthermore, we uncovered that Gbetagamma can both translocate Csk to the plasma membrane and directly increase Csk kinase activity. Our genetic and biochemical studies demonstrate that Csk plays a critical role in mediating G protein signals to actin cytoskeletal reorganization.

Identification of TMEM230 mutations in familial Parkinson's disease

Parkinsons disease is the second most common neurodegenerative disorder without effective treatment. It is generally sporadic with unknown etiology. However, genetic studies of rare familial forms have led to the identification of mutations in several genes, which are linked to typical Parkinsons disease or parkinsonian disorders. The pathogenesis of Parkinsons disease remains largely elusive. Here we report a locus for autosomal dominant, clinically typical and Lewy body–confirmed Parkinsons disease on the short arm of chromosome 20 (20pter-p12) and identify TMEM230 as the disease-causing gene. We show that TMEM230 encodes a transmembrane protein of secretory/recycling vesicles, including synaptic vesicles in neurons. Disease-linked TMEM230 mutants impair synaptic vesicle trafficking. Our data provide genetic evidence that a mutant transmembrane protein of synaptic vesicles in neurons is etiologically linked to Parkinsons disease, with implications for understanding the pathogenic mechanism of Parkinsons disease and for developing rational therapies.

SnapShot-Seq: A Method for Extracting Genome-Wide, In Vivo mRNA Dynamics from a Single Total RNA Sample

mRNA synthesis, processing, and destruction involve a complex series of molecular steps that are incompletely understood. Because the RNA intermediates in each of these steps have finite lifetimes, extensive mechanistic and dynamical information is encoded in total cellular RNA. Here we report the development of SnapShot-Seq, a set of computational methods that allow the determination of in vivo rates of pre-mRNA synthesis, splicing, intron degradation, and mRNA decay from a single RNA-Seq snapshot of total cellular RNA. SnapShot-Seq can detect in vivo changes in the rates of specific steps of splicing, and it provides genome-wide estimates of pre-mRNA synthesis rates comparable to those obtained via labeling of newly synthesized RNA. We used SnapShot-Seq to investigate the origins of the intrinsic bimodality of metazoan gene expression levels, and our results suggest that this bimodality is partly due to spillover of transcriptional activation from highly expressed genes to their poorly expressed neighbors. SnapShot-Seq dramatically expands the information obtainable from a standard RNA-Seq experiment.

Novel regulation and function of Src tyrosine kinase.

Abstract. Src tyrosine kinase is a critical signal transducer that modulates a wide variety of cellular functions. Misregulation of Src leads to cell transformation and cancer. Heterotrimeric guanine-nucleotide-binding proteins (G proteins) are another group of signaling molecules that transduce signals from cell-surface receptors to generate physiological responses. Recently, it was discovered that Gαs and Gαi could directly stimulate Src family tyrosine kinase activity. This novel regulation of Src tyrosine kinase by G proteins provides insights into the adenylyl cyclase-independent signaling mechanisms involved in ligand-induced receptor desensitization, internalization and other physiological processes.

GSK3 temporally regulates neurogenin 2 proneural activity in the neocortex.

The neocortex is comprised of six neuronal layers that are generated in a defined temporal sequence. While extrinsic and intrinsic cues are known to regulate the sequential production of neocortical neurons, how these factors interact and function in a coordinated manner is poorly understood. The proneural gene Neurog2 is expressed in progenitors throughout corticogenesis, but is only required to specify early-born, deep-layer neuronal identities. Here, we examined how neuronal differentiation in general and Neurog2 function in particular are temporally controlled during murine neocortical development. We found that Neurog2 proneural activity declines in late corticogenesis, correlating with its phosphorylation by GSK3 kinase. Accordingly, GSK3 activity, which is negatively regulated by canonical Wnt signaling, increases over developmental time, while Wnt signaling correspondingly decreases. When ectopically activated, GSK3 inhibits Neurog2-mediated transcription in cultured cells and Neurog2 proneural activities in vivo. Conversely, a reduction in GSK3 activity promotes the precocious differentiation of later stage cortical progenitors without influencing laminar fate specification. Mechanistically, we show that GSK3 suppresses Neurog2 activity by influencing its choice of dimerization partner, promoting heterodimeric interactions with E47 (Tcfe2a), as opposed to Neurog2–Neurog2 homodimer formation, which occurs when GSK3 activity levels are low. At the functional level, Neurog2–E47 heterodimers have a reduced ability to transactivate neuronal differentiation genes compared with Neurog2–Neurog2 homodimers, both in vitro and in vivo. We thus conclude that the temporal regulation of Neurog2–E47 heterodimerization by GSK3 is a central component of the neuronal differentiation “clock” that coordinates the timing and tempo of neocortical neurogenesis in mouse.

Novel Signaling Pathway Through the β-Adrenergic Receptor

Abstract β -Adrenergic receptors transduce signals through the heterotrimeric G protein Gαs to regulate cardiac function. Recently, it has been discovered that Gαs could directly stimulate the Src family tyrosine kinase activity. This novel signaling pathway provides insights into the cAMP–PKA-independent signaling mechanisms used by β -adrenergic receptors to regulate apoptosis, receptor desensitization, and other physiological functions.