Phosphoproteomics data classify hematological cancer cell lines according to tumor type and sensitivity to kinase inhibitors

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Disulfide-activated protein kinase G Iα regulates cardiac diastolic relaxation and fine-tunes the Frank-Starling response.

The Frank-Starling mechanism allows the amount of blood entering the heart from the veins to be precisely matched with the amount pumped out to the arterial circulation. As the heart fills with blood during diastole, the myocardium is stretched and oxidants are produced. Here we show that protein kinase G Iα (PKGIα) is oxidant-activated during stretch and this form of the kinase selectively phosphorylates cardiac phospholamban Ser16-a site important for diastolic relaxation. We find that hearts of Cys42Ser PKGIα knock-in (KI) mice, which are resistant to PKGIα oxidation, have diastolic dysfunction and a diminished ability to couple ventricular filling with cardiac output on a beat-to-beat basis. Intracellular calcium dynamics of ventricular myocytes isolated from KI hearts are altered in a manner consistent with impaired relaxation and contractile function. We conclude that oxidation of PKGIα during myocardial stretch is crucial for diastolic relaxation and fine-tunes the Frank-Starling response

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

Stabilization of the metaphase spindle by Cdc14 is required for recombinational DNA repair

This work was funded by grants from the Ministerio de Economía y Competitividad (BFU2013-41216-P) and the Ramón y Cajal Programme (RYC-201210363) conceded to A.C-B. F.R. was recipient of a fellowship from Ministerio de Economía y Competitividad

Kinase-substrate enrichment analysis provides insights into the heterogeneity of signaling pathway activation in leukemia cells

Kinases determine the phenotypes of many cancer cells, but the frequency with which individual kinases are activated in primary tumors remains largely unknown. We used a computational approach, termed kinase-substrate enrichment analysis (KSEA), to systematically infer the activation of given kinase pathways from mass spectrometry-based phosphoproteomic analysis of acute myeloid leukemia (AML) cells. Experiments conducted in cell lines validated the approach and, furthermore, revealed that DNAdependent protein kinase (DNA-PK) was activated as a result of inhibiting the phosphoinositide 3-kinase (PI3K)-mammalian target of rapamycin (mTOR) signaling pathway. Application of KSEA to primary AML cells identified PI3K, casein kinases (CKs), cyclin-dependent kinases (CDKs), and p21-activated kinases (PAKs) as the kinase substrate groups most frequently enriched in this cancer type. Substrates phosphorylated by extracellular signal-regulated kinase (ERK) and cell division cycle 7 (CDC7) were enriched in primary AML cells that were resistant to inhibition of PI3K-mTOR signaling, whereas substrates of the kinases Abl, Lck, Src, and CDK1 were increased in abundance in inhibitor-sensitive cells. Modeling based on the abundances of these substrate groups accurately predicted sensitivity to a dual PI3K and mTOR inhibitor in two independent sets of primary AML cells isolated from patients. Thus, our study demonstrates KSEA as an untargeted method for the systematic profiling of kinase pathway activities and for increasing our understanding of diseases caused by the dysregulation of signaling pathways. Copyright 2008 by the American Association for the Advancement of Scienc

Class IA phosphoinositide 3-kinases are obligate p85-p110 heterodimers

Class IA phosphoinositide 3-kinases (PI3Ks) signal downstream of tyrosine kinases and Ras and control a wide variety of biological responses. In mammals, these heterodimeric PI3Ks consist of a p110 catalytic subunit (p110α, p110β, or p110δ) bound to any of five distinct regulatory subunits (p85α, p85β, p55γ, p55α, and p50α, collectively referred to as “p85s”). The relative expression levels of p85 and p110 have been invoked to explain key features of PI3K signaling. For example, free (i.e., non-p110-bound) p85α has been proposed to negatively regulate PI3K signaling by competition with p85/p110 for recruitment to phosphotyrosine docking sites. Using affinity and ion exchange chromatography and quantitative mass spectrometry, we demonstrate that the p85 and p110 subunits are present in equimolar amounts in mammalian cell lines and tissues. No evidence for free p85 or p110 subunits could be obtained. Cell lines contain 10,000–15,000 p85/p110 complexes per cell, with p110β and p110δ being the most prevalent catalytic subunits in nonleukocytes and leukocytes, respectively. These results argue against a role of free p85 in PI3K signaling and provide insights into the nonredundant functions of the different class IA PI3K isoforms