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y the intracellular AMP ATP ratio, but also by phosphorylation at Thr by AMPK kinases . Recently two AMPKK's happen to be identified, namely LKB and CaMKK . In the heart, AMPK could be activated for the duration of exercise, hypoxia and ischemia . The key downstream target of AMPK is acetyl CoA carboxylase . Active AMPK phosphorylates Ubiquitin conjugation inhibitor ACC at Ser thereby inactivating ACC which final results in an increase in LCFA oxidation. AMPK is a protein consisting of three distinct subunits, the catalytic subunit and also the regulatory and γ subunits. Although two isoforms from the catalytic subunit are present in the heart, the subunit is predominant . Recently, it was shown that in heart from transgenic mice overexpressing a dominant negative AMPK mutant, contraction was nonetheless able to stimulate glucose uptake .
This demonstrates that contraction induced glucose uptake can only be partly ascribed to AMPK. Interestingly, in H K skeletal muscle cells expressing dominant negative AMPK , Ubiquitin conjugation inhibitor a cellpermeable diacylglycerol analogue, phorbol myristate acetate , was able to stimulate glucose uptake , suggesting that a protein kinase sensitive to DAG is involved. In L skeletal muscle cells it has been demonstrated that the DAG sensitive protein kinase D directly contributes to basal glucose uptake . Taken together, these final results suggest that PKD, in addition to AMPK, could also mediate contraction induced glucose Docetaxel uptake. Previously, PKD has been classified as a member from the PKC loved ones , and has been frequently referred to as PKC . The PKC loved ones consists of three subfamilies, i.e standard, novel and atypical PKCs .
Standard PKCs require diacylglycerol and Ca for their activation, whereas novel PKCs VEGF also require DAG but are Ca independent, and atypical PKC's require neither DAG nor calcium . PKD possesses a DAG binding site, and was therefore subclassified Docetaxel as a novel PKC isoform, i.e PKC . Nevertheless, the catalytic domain of PKD is much more closely related to that from the Ca calmodulin regulated protein kinases and displays reasonably small homology towards the catalytic domains from the PKC loved ones . Furthermore, in comparison to other members from the PKC loved ones, PKD possesses an further pleckstrin homology domain, a putative transmembrane sequence and lacks a pseudosubstrate region.
For that reason, PKD has been positioned into a novel kinase loved ones, comprising three members: PKD , PKD and PKD In non stimulated mammalian cell lines, PKD was identified to be localized towards the cytosol and many intracellular membrane compartments such as Golgi and mitochondria . Treatment of COS cells with phorbol esters Conjugating enzyme inhibitor induced a persistent translocation of PKD from the cytosol towards the plasma membrane, requiring the DAG binding domain. In addition to phorbol esters, PKD may also be activated by different agonists, most of which bind to G protein coupled receptors . GPCR mediated activation of PKD is mediated by members from the PKC loved ones, and involves a phosphorylation of two serine residues within the activation loop, i.e Ser and Ser . In addition to the transphosphorylation at Ser , PKD is autophosphorylated at Ser upon activation . Ser autophosphorylation has also been shown to occur upon phorbol ester stimulation, and was identified to correlate accurately with catalytic activity of PKD .
PKD has been identified to be present in the heart, where it is also activated by phorbol ester treatment . In addition, GPCRs happen to be shown Docetaxel to activate PKD in the heart through a PKC dependent mechanism . The heart expresses many standard and novel PKC isoforms . It has not however been investigated which of these PKCs is involved in GPCR mediated PKD activation. In the present study, we explored in cardiac myocytes whether or not PKD is activated by contraction, and whether or not this is linked to glucose uptake. 1st, we determined whether or not electrically induced contraction and treatment of cardiac myocytes with oligomycin stimulated PKD translocation, Ser phosphorylation, as well as PKD enzymatic activity.
Subsequently, the positioning of PKD relative to AMPK was studied with in vitro kinase assays Docetaxel and in cardiac myocytes isolated from AMPK ? ? mice. Thereafter, we attempted to determine upstream kinases involved in oligomycin contractioninduced PKD activation in cardiac myocytes. Finally, we linked contraction induced PKD activation to contraction induced glucose uptake by using pharmacological agents that inhibit selected PKCs as well as PKD. The combined observations reveal that PKD is activated in cardiac myocytes by contraction, independent of AMPK activation. This suggests that there is a PKD mediated contraction signaling pathway top to GLUT translocation, parallel to AMPK signaling. Autophosphorylation of PKD at Ser is regarded as to be an accurate indicator of activity of this protein kinase . We 1st determined the optimal conditions for oligomycin treatment of cardiac myocytes . Treatment of cardiac myocytes with oligomycin at M already improved Ser phosphorylation by . fold, which slightly improved to . fold abo