"Another interesting kinase predicted by our microarray results to act downstream of HGF/c-Met stimulation is the protein kinase A catalytic subunit-beta (PRKACB). To determine whether the activity of PKA is enhanced in response to c-Met activation, we examined PKA-dependent phosphorylation events in cells using an antibody that recognizes phosphorylated PKA substrates 
. We observed that PKA-dependent phosphorylation was substantially enhanced upon HGF treatment, while the total protein level of PKA was unchanged (Fig. 4f). "
[Show abstract][Hide abstract] ABSTRACT: Protein phosphorylation is a dynamic and reversible event that greatly influences cellular function. Identifying the key regulatory elements that determine cellular phenotypes during development and oncogenesis requires the ability to dynamically monitor proteome-wide events. Here, we report the development of a new strategy to monitor dynamic changes of protein phosphorylation in cells and tissues using functional protein microarrays as the readout. To demonstrate this technology's ability to identify condition-dependent phosphorylation events, human protein microarrays were incubated with lysates from cells or tissues under activation or inhibition of c-Met, a receptor tyrosine kinase involved in tissue morphogenesis and malignancy. By comparing the differences between the protein phosphorylation profiles obtained using the protein microarrays, we were able to recover many of the proteins that are known to be specifically activated (i.e., phosphorylated) upon c-Met activation by the hepatocyte growth factor (HGF). Most importantly, we discovered many proteins that were differentially phosphorylated by lysates from cells or tissues when the c-Met pathway was active. Using phosphorylation-specific antibodies, we were able to validate several candidate proteins as new downstream components of the c-Met signaling pathway in cells. We envision that this new approach, like its DNA microarray counterpart, can be further extended toward profiling dynamics of global protein phosphorylation under many different physiological conditions both in cellulo and in vivo in a high-throughput and cost-effective fashion.
PLoS ONE 09/2013; 8(9):e72671. DOI:10.1371/journal.pone.0072671 · 3.23 Impact Factor
"Since phosphorylation of ribosomal S6 is thought to be specific to TORC1 signalling, we decided to look further into its phosphorylation. It has previously been demonstrated that the anti-PAS antibody, which recognises phosphorylation of the [R/ K]X[R/K]XX[S/T] consensus motif in mammalian S6K1 and other AGC kinase substrates (Manning et al., 2002; Pearson and Kemp, 1991) (Cell Signalling Technology), detects phosphorylation of fission yeast Rps6 serine 235 (Nakashima et al., 2010). Ribosomal S6 is encoded by two genes in fission yeast: rps601 and rps602. "
[Show abstract][Hide abstract] ABSTRACT: TOR (Target Of Rapamycin) signalling coordinates cell growth and division in response to changes in the nutritional environment of the cell. TOR kinases form two distinct complexes: TORC1 and TORC2. In mammals, the TORC1 controlled S6K1 kinase phosphorylates the ribosomal protein S6 thereby co-ordinating cell size and nutritional status. We show that the Schizosaccharomyces pombe AGC kinase Gad8 co-immunoprecipitates with the ribosomal protein S6 (Rps6) and regulates its phosphorylation status. It has previously been shown that Gad8 is phosphorylated by TORC2. Consistent with this, we find that TORC2 as well as TORC1 modulates Rps6 phosphorylation. Therefore, S6 phosphorylation in fission yeast actually represents a read-out of the combined activities of TORC1 and TORC2. In contrast, we find that the in vivo phosphorylation status of Maf1 (a repressor of RNA polymerase III) specifically correlates with TORC1 activity.
Biology Open 09/2012; 1(9):884-8. DOI:10.1242/bio.20122022 · 2.42 Impact Factor
"via ADP/ATP [20, 21], 3,5-diiodothyronine , palmitate , and calcium ; and (iii) by reversible phosphorylation of the subunits [25–28]. Based on consensus sequences for protein kinase A (PKA) , 53 potential phosphorylation sites for serine or threonine are present in the bovine heart enzyme (11 in subunit I, "
[Show abstract][Hide abstract] ABSTRACT: Cytochrome c oxidase (COX), the terminal enzyme of the mitochondrial electron transport chain, is regulated by isozyme expression, allosteric effectors such as the ATP/ADP ratio, and reversible phosphorylation. Of particular interest is the "allosteric ATP-inhibition," which has been hypothesized to keep the mitochondrial membrane potential at low healthy values (<140 mV), thus preventing the formation of superoxide radical anions, which have been implicated in multiple degenerative diseases. It has been proposed that the "allosteric ATP-inhibition" is switched on by the protein kinase A-dependent phosphorylation of COX. The goal of this study was to identify the phosphorylation site(s) involved in the "allosteric ATP-inhibition" of COX. We report the mass spectrometric identification of four new phosphorylation sites in bovine heart COX. The identified phosphorylation sites include Tyr-218 in subunit II, Ser-1 in subunit Va, Ser-2 in subunit Vb, and Ser-1 in subunit VIIc. With the exception of Ser-2 in subunit Vb, the identified phosphorylation sites were found in enzyme samples with and without "allosteric ATP inhibition," making Ser-2 of subunit Vb a candidate site enabling allosteric regulation. We therefore hypothesize that additional phosphorylation(s) may be required for the "allosteric ATP-inhibition," and that these sites may be easily dephosphorylated or difficult to identify by mass spectrometry.
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