Protein kinase A | EPFL Graph Search

In cell biology, protein kinase A (PKA) is a family of serine-threonine kinase whose activity is dependent on cellular levels of cyclic AMP (cAMP). PKA is also known as cAMP-dependent protein kinase (). PKA has several functions in the cell, including regulation of glycogen, sugar, and lipid metabolism. It should not be confused with 5'-AMP-activated protein kinase (AMP-activated protein kinase). History Protein kinase A, more precisely known as adenosine 3',5'-monophosphate (cyclic AMP)-dependent protein kinase, abbreviated to PKA, was discovered by chemists Edmond H. Fischer and Edwin G. Krebs in 1968. They won the Nobel Prize in Physiology or Medicine in 1992 for their work on phosphorylation and dephosphorylation and how it relates to PKA activity. PKA is one of the most widely researched protein kinases, in part because of its uniqueness; out of 540 different protein kinase genes that make up the human kinome, only one other protein kinase, casein ki

Physiological regulation of the CDK16/PCTAIRE-1 protein kinase and its proposed role in the brain

Saifeldin Nasser Mohamed Ibrahim Shehata

Cell signalling, mediated to a large extent by protein kinase phosphorylation, plays a vital role in regulation of cellular function. PCTAIRE-1 (also known as cyclin-dependent protein kinase (CDK)16), is a Ser/Thr kinase that has been implicated in many cellular processes, including cell cycle, apoptosis, insulin secretion, spermatogenesis, neurite outgrowth, and vesicle trafficking. Most recently, it has been proposed as a novel X-linked intellectual disability (XLID) gene, where loss-of-function mutations have been found in patients. The precise molecular mechanisms that regulate PCTAIRE-1 remained largely obscure, and only few substrates have been proposed with no clear functional significance and physiological role. To understand the function of PCTAIRE-1, we previously utilised a peptide library screen to determine the consensus phosphorylation motif. We showed that PCTAIRE-1 preferentially phosphorylated peptide motifs that differed from the classical CDK family substrate preference, suggesting a more distinct role for the kinase. Furthermore, we showed that cyclin Y, a novel cyclin, robustly binds and activates PCTAIRE-1 > 100-fold. In this thesis, we have identified two phosphorylation sites on cyclin Y that are essential for binding the well-known adaptor protein 14-3-3, which we propose stabilizes cyclin Y in a favourable PCTAIRE-1-binding conformation. Mutation of these sites to non-phosphorylatable Ser residues abolished PCTAIRE-1 binding and activation. Furthermore, we have cloned human PCTAIRE-1 mutants identified in XLID patients, and confirmed their failure to bind the cyclin Y-14-3-3 activating complex. In order to understand the physiological relevance of PCTAIRE-1 activity, we have utilised a chemical genetics approach that exploits the ability of an engineered PCTAIRE-1 mutant to selectively modify its substrates, allowing them to be uniquely purified. We have identified three PCTAIRE-1 substrates (AAK1, dynamin 1 and synaptojanin 1) in mouse brain that regulate crucial steps of receptor endocytosis, namely receptor binding, vesicle scission and vesicle uncoating, which are all involved in the regulation of neuronal synaptic transmission. Collectively, this thesis work has provided key molecular regulatory mechanisms and potential downstream targets of PCTAIRE-1, which has laid the foundations for future studies of the role that PCTAIRE-1 plays in specific cellular functions and physiological and pathological settings.

EPFL2017

Identification and functional characterization of protein kinases that modulate Notch signaling under physiological conditions and in cancer

Chhavi Jain

The Notch signaling pathway is a key regulator of cell fate decisions in embryonic development and in adult tissue homeostasis. Mounting evidence suggests that Notch signaling is frequently deregulated in human neoplasms, where depending upon the cellular context it can function both as an oncogene or a tumor suppressor. A plethora of studies shed light on how Notch crosstalks with signaling pathways downstream to manifest either its oncogenic or tumor suppressive activity. However, regulatory networks upstream of the Notch signaling pathway are still poorly understood. Since protein kinases are well-known regulators of a majority of cell signaling pathways, the aim of the current study was to identify novel positive and negative regulatory kinases that modulate Notch signaling. Using a HeLa cell based co-culture assay to read Notch-dependent luciferase activity, a small interference RNA (siRNA) library against the human kinase genome was previously tested in a high-throughput manner. Validation of top candidate kinases from the screening using both siRNA as well as pharmacological inhibitors led to further elimination of false positives and identification of Protein Kinase B (AKT2) and Calmodulin Kinase II (CaMKII) as key positive while Janus kinases (JAKs) as key negative regulators of the Notch signaling pathway. In the first part of the study, we analyzed the positive regulation of AKT2 and CaMKII kinases on Notch signaling in the T-cell acute lymphoblastic leukemia (T-ALL) cells where Notch is oncogenic. It was shown that pharmacological inhibition of either AKT2 or CaMKII kinase in T-ALL cell lines in vitro abrogated the expression of active Notch1 intracellular domain (N1-ICD) as well as of Notch target genes. Moreover, inhibition of these kinases blocked proliferation of T-ALL cells. In the second part of the study, we analyzed the negative regulation of JAK kinases on Notch signaling using a distinct physiological context where Notch is tumor suppressive. It was shown that pharmacological inhibition of JAK kinases led to an induction of Notch receptor transcription and of Notch target genes in the human primary epidermal keratinocytes (HPEK) as well as in the skin squamous cell carcinoma (SCC) cell lines. In addition, the pharmacological inhibition of JAK kinases induced a block in proliferation, cell cycle arrest and differentiation in HPEKs and skin SCCs by increased expression of early differentiation markers. Suppression of Notch signaling in primary keratinocytes counteracted the differentiation-inducing effect of JAK inhibition. Since JAK inhibition affected Notch transcription, global gene expression profiling using RNA sequencing was done to identify transcription factors involved in an indirect regulation of JAK kinases on the Notch cascade. EGR1 and EGR2 belonging to the early growth response family of transcription factors were significantly modulated downstream of JAK inhibition. In vivo, topical inhibition of JAK kinases provided marked resistance against chemically induced cutaneous carcinogenesis.Taken together, our data reveals a key role of AKT2 and CaMKII kinases in positive regulation while of JAK kinases in the negative regulation of Notch signaling, of potential relevance for combinatory approaches for cancer therapy. Pharmacological inhibitors against these kinases could be tested for their ability to modulate Notch signaling and may prove highly beneficial in the therapy of Notch-driven cancers.

EPFL2016

CDK4 Phosphorylates AMPK alpha 2 to Inhibit Its Activity and Repress Fatty Acid Oxidation

Caterina Collodet, Anita Nasrallah

The roles of CDK4 in the cell cycle have been extensively studied, but less is known about the mechanisms underlying the metabolic regulation by CDK4. Here, we report that CDK4 promotes anaerobic glycolysis and represses fatty acid oxidation in mouse embryonic fibroblasts (MEFs) by targeting the AMP- activated protein kinase (AMPK). We also show that fatty acid oxidation (FAO) is specifically induced by AMPK complexes containing the alpha 2 subunit. Moreover, we report that CDK4 represses FAO through direct phosphorylation and inhibition of AMPK alpha 2. The expression of non- phosphorylatable AMPK alpha 2 mutants, or the use of a CDK4 inhibitor, increased FAO rates in MEFs and myotubes. In addition, Cdk4(-/-) mice have increased oxidative metabolism and exercise capacity. Inhibition of CDK4 mimicked these alterations in normal mice, but not when skeletal muscle was AMPK deficient. This novel mechanism explains how CDK4 promotes anabolism by blocking catabolic processes (FAO) that are activated by AMPK.

Cell Press2017