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It is well documented that unliganded thyroid hormone receptor (TR) functions as a transcriptional repressor of specific cellular target genes by acting in concert with a corepressor complex harboring histone deacetylase (HDAC) activity. To fully explore the cofactors that interact with the transcriptionally repressive form of TR, we biochemically isolated a multiprotein complex that assembles on a TR-retinoid X receptor (RXR) heterodimer in HeLa nuclear extracts and identified its polypeptide components by mass spectrometry. A subset of TR-RXR-associated polypeptides included NCoR, SMRT, TBL1, and HDAC3, which represent the core components of a previously described NCoR/SMRT corepressor complex. We also identified several polypeptides that constitute a DNA-dependent protein kinase (DNA-PK) enzyme complex, a regulator of DNA repair, recombination, and transcription. These polypeptides included the catalytic subunit DNA-PKcs, the regulatory subunits Ku70 and Ku86, and the poly(ADP-ribose) polymerase 1. Density gradient fractionation and immunoprecipitation analyses provided evidence for the existence of a high molecular weight TR-RXR-corepressor holocomplex containing both NCoR/SMRT and DNA-PK complexes. Chromatin immunoprecipitation studies confirmed that unliganded TR-RXR recruits both complexes to the triiodothyronine-responsive region of growth hormone gene in vivo. Interestingly, DNA-PKcs, a member of the phosphatidylinositol 3-kinase family, was found to phosphorylate HDAC3 when the purified TR-RXR-corepressor holocomplex was incubated with ATP. This phosphorylation was accompanied by a significant enhancement of the HDAC activity of this complex. Collectively, our results indicated that DNA-PK promotes the establishment of a repressive chromatin at a TR target promoter by enhancing the HDAC activity of the receptor-bound NCoR/SMRT corepressor complex. [ABSTRACT FROM AUTHOR]

Activation of members of the protein kinase AGC (cAMP dependent, cGMP dependent, and protein kinase C) family is regulated primarily by phosphorylation at two sites: a conserved threonine residue in the activation loop and a serine/threonine residue in a hydrophobic motif (HM) near the COOH terminus. Although phosphorylation of these kinases in the activation loop has been found to be mediated by phosphoinositide-dependent protein kinase-1 (PDK1), the kinase(s) that catalyzes AGC kinase phosphorylation in the HM remains uncharacterized. So far, at least 10 kinases have been suggested to function as an HM kinase or the so-called ‘PDK2,’ including mitogen-activated protein (MAP) kinase-activated protein kinase-2 (MK2), integrin-linked kinase (ILK), p38 MAP kinase, protein kinase Cα (PKCα), PKCβ, the NIMA-related kinase-6 (NEK6), the mammalian target of rapamycin (mTOR), the double-stranded DNA-dependent protein kinase (DNK-PK), and the ataxia telangiectasia mutated (ATM) gene product. However, whether any or all of these kinases act as a physiological HM kinase remains to be established. Nonetheless, available data suggest that multiple systems may be used in cells to regulate the activation of the AGC family kinases. It is possible that, unlike activation loop phosphorylation, phosphorylation of the HM site in the different AGC family kinases is mediated by distinct kinases. In addition, phosphorylation of the AGC family kinase at the HM site could be cell type, signaling pathway, and substrate specific. Identification and characterization of the bonafide HM kinase(s) will be essential to verify these hypotheses. [ABSTRACT FROM AUTHOR]

Birukova, Anna A., Feng Liu, Joe G. N. Garcia, and Alexander D. Verin. Protein kinase A attenuates endothelial cell barrier dysfunction induced by microtubule disassembly. Am J Physiol Lung Cell Mol Physiol 287: L86-L93, 2004. First published March 5, 2004; 10.1152/ajplung.00441.2003.&ndsah;Cross between the actin cytoskeleton and the microyubule (MT) network plays a critical role in regulation of endothelial permeability. We have previously demonstrated that MT disruption by nocodazole results in increases in MLC phospholrylation, cardinal features of endothelial barrier dysfunction (Verin AD, Birukova A, Wang P, Liu F, Kaibuchi K, Ricks-cord A, Natarajan V, Alieva A, Garcia JG, and Verin AD. J Cell Physiol. In press.). Although activation of PKA opposes barrier-disrupting effects of edemagenic agents on confluent EC monolayers, information about the molecular mechanisms of PKA-mediated EC barrier protection is limited. Our results suggest that MT disassembly alters neither intracellular cAMP levels and PKA enzymatic activity; however, elevation of cAMP levels and PKA activation by either cholera toxin or forskolin dramatically attenuates the decline in transendothelial electrical resistance induced by nocodazole in human pulmonary EC. Barrier-protective effects of PKA on EC were associated with PKA-mediated inhibition of nocodazole-induced stress fiber formation, Rho activation, phosporylyzation of myosin phosphatase regulatory subunit at Thr696, and decreased MLC phosphorylation. In addition, forskolin pretreatment attenuated MT disassembly induced by nocodazole. These results suggest a critical role for PKA activity in stabilization of MT cytoskeleton and provide a novel mechanism for cAMP-mediated regu;ation of Rho-induced actin cytoskeletal remodeling, actomyosin contraction, and EC barrier dysfunction induced by MT disassembly. [ABSTRACT FROM AUTHOR]

Studies the regulation of cyclin-dependent kinase 5 (Cdk5) and casein kinase 1 (CK1) by metabotropic glutamate receptors (mGluRs). Effect of group 1 mGluR agonist on phosphorylation of DARPP-32 at Thr-75 and Ser-137; Effect of group 1 mGluR agonist on Cdk5 and CK1 activities; Effect of Cdk5 and CK1 inhibitors on phosphorylation of DARPP-32 at Thr-75 and Ser-137.

3-Phosphoinositide-dependent protein kinase-1 (PDK1) mediates phosphorylation and activation of members of the AGC protein kinase family and plays an essential role in insulin signaling and action. However, whether and how PDK1 activity is regulated in cells remains largely uncharacterized. In the present study, we show that PDK1 undergoes insulin-stimulated and phosphatidylinositol 3-kinase-dependent phosphorylation at Ser244 in the activation loop and at a novel site: Ser163 in the hinge region between the two lobes of the kinase domain. Sequence alignment studies revealed that the residue corresponding to Ser163 of PDK1 in all other AGC kinases is glutamate, suggesting that a negative charge at this site may be important for PDK1 function. Replacing Ser163 with a negatively charged residue, glutamate, led to a 2-fold increase in PDK1 activity. Molecular modeling studies suggested that phosphorylated Ser163 may form additional hydrogen bonds with Tyr149 and Gln223. In support of this, mutation of Tyr149 to Ala is sufficient to reduce PDK1 activity. Taken together, our results suggest that PDK1 phosphorylation of Ser163 may provide a mechanism to fine-tune PDK1 activity and function in cells. [ABSTRACT FROM AUTHOR]

Src tyrosine kinase suppresses KCNQ (M-type) K+ channels in a subunit-specific manner representing a mode of modulation distinct from that involving G protein-coupled receptors. We probed the molecular and biophysical mechanisms of this modulation using mutagenesis, biochemistry, and both whole-cell and single channel modes of patch clamp recording. Immunoprecipitation assays showed that Src associates with KCNQ2-5 subunits but phosphorylates only KCNQ3-5. Using KCNQ3 as a background, we found that mutation of a tyrosine in the amino terminus (Tyr-67) or one in the carboxyl terminus (Tyr-349) abolished Src-dependent modulation of heterologously expressed KCNQ2/3 heteromultimers. The tyrosine phosphorylation was much weaker for either the KCNQ3-Y67F or KCNQ3-Y349F mutants and wholly absent in the KCNQ3-Y67F/Y349F double mutant. Biotinylation assays showed that Src activity does not alter the membrane abundance of channels in the plasma membrane. In recordings from cell-attached patches containing a single KCNQ2/3 channel, we found that Src inhibits the open probability of the channels. Kinetic analysis was consistent with the channels having two discrete open times and three closed times. Src activity reduced the durations of the longest open time and lengthened the longest closed time of the channels. The implications for the mechanisms of channel regulation by the dual phosphorylations on both channel termini are discussed. [ABSTRACT FROM AUTHOR]

Activation of mouse 3-phosphoinositide-dependent protein kinase-1 (mPDK1) requires phosphorylation at a conserved serine residue, Ser[sup 244], in the activation loop. However, the mechanism by which mPDK1 is phosphorylated at this site remains unclear. We have found that kinase-defective mPDK1 (mPDK1[sup KD]), but not a kinasedefective mPDK1 in which Ser[sup 244] was replaced with alanine (mPDK1[sup KD/S244A]), is significantly phosphorylated in intact cells and is a direct substrate of wild-type mPDK1 fused to the yellow fluorescence protein. Phosphoamino acid analysis and phosphopeptide mapping studies revealed that mPDK1 trans-autophosphorylation occurred mainly on Ser[sup 244]. On the other hand, Ser[sup 399] and Thr[sup 516], two recently identified autophosphorylation sites of mPDK1, are phosphorylated primarily through a cis mechanism. In vivo labeling studies revealed that insulin stimulated both mPDK1[sup KD] and mPDK1[sup KD/S244A] phosphorylation in Chinese hamster ovary cells overexpressing the insulin receptor. However, Western blot analysis using a phosphospecific antibody revealed no increase in insulin-stimulated phosphorylation of Ser[sup 244] in these cells overexpressing mPDK1, mPDK1 undergoes dimerization in cells and this self-association is enhanced by kinase inactivation. Deletion of the extreme C terminus disrupts mPDK1 dimerization and Ser[sup 244] trans-phosphorylation, suggesting that dimerization is important for mPDK1 transphosphorylation. Taken together, our results show that mPDK1 autophosphorylation occurs at multiple sites through both cis and trans mechanisms and suggest that dimerization and trans-phosphorylation may serve as mechanisms to regulate PDK1 activity in cells. [ABSTRACT FROM AUTHOR]

Activation of the AMP-activated protein kinase (AMPK) mediates adiponectin signalling to regulate glucose and lipid metabolism in muscle. However, the molecular mechanism by which adiponectin activates AMPK remains largely unknown. We have recently found that the BAR, PH, and PTB domain-containing adaptor protein APPL1 binds directly to the intracellular part of adiponectin receptors and mediates adiponectin-stimulated AMPK activation (Mao et al. (2006) Nat Cell Biol. 8,516-523). Here we report that APPL1 directly binds to LKB1, an upstream kinase of AMPK, via the BAR domain. Under basal condition, LKBI is localized predominantly in the nucleus of C2C12 cells. Treating cells with either adiponectin or metformin significantly stimulates LKB1 translocation to the cytosol. Overexpression of LKB1SL-26, a mutant form of LKB1 which maintains intact kinase activity but localizes exclusively in the nucleus, inhibited adiponectin and metformin-stimulated AMPK activation in C2C12 cells. The regulation of LKB1 translocation is dependent on phosphorylation at Ser307, which is mediated by protein kinase Cz (PKCz). Taken together, our results reveal a novel mechanism by which adiponectin and metformin stimulate LKB1 cytosolic translocation, which is essential for AMPK activation in muscle cells. ADA-Funded Research [ABSTRACT FROM AUTHOR]