KRSA: An R package and R Shiny web application for an end-to-end upstream kinase analysis of kinome array data

Phosphorylation by serine-threonine and tyrosine kinases is critical for determining protein function. Array-based platforms for measuring reporter peptide signal levels allow for differential phosphorylation analysis between conditions for distinct active kinases. Peptide array technologies like the PamStation12 from PamGene allow for generating high-throughput, multi-dimensional, and complex functional proteomics data. As the adoption rate of such technologies increases, there is an imperative need for software tools that streamline the process of analyzing such data. We present Kinome Random Sampling Analyzer (KRSA), an R package and R Shiny web-application for analyzing kinome array data to help users better understand the patterns of functional proteomics in complex biological systems. KRSA is an All-In-One tool that reads, formats, fits models, analyzes, and visualizes PamStation12 kinome data. While the underlying algorithm has been experimentally validated in previous publications, we demonstrate KRSA workflow on dorsolateral prefrontal cortex (DLPFC) in male (n = 3) and female (n = 3) subjects to identify differential phosphorylation signatures and upstream kinase activity. Kinase activity differences between males and females were compared to a previously published kinome dataset (11 female and 7 male subjects) which showed similar global phosphorylation signals patterns.

A stop or go switch: glycogen synthase kinase 3β phosphorylation of the kinesin 1 motor domain at Ser314 halts motility without detaching from microtubules

ABSTRACT It is more than 25 years since the discovery that kinesin 1 is phosphorylated by several protein kinases. However, fundamental questions still remain as to how specific protein kinase(s) contribute to particular motor functions under physiological conditions. Because, within an whole organism, kinase cascades display considerable crosstalk and play multiple roles in cell homeostasis, deciphering which kinase(s) is/are involved in a particular process has been challenging. Previously, we found that GSK3β plays a role in motor function. Here, we report that a particular site on kinesin 1 motor domain (KHC), S314, is phosphorylated by GSK3β in vivo. The GSK3β-phosphomimetic-KHCS314D stalled kinesin 1 motility without dissociating from microtubules, indicating that constitutive GSK3β phosphorylation of the motor domain acts as a STOP. In contrast, uncoordinated mitochondrial motility was observed in CRISPR/Cas9-GSK3β non-phosphorylatable-KHCS314A Drosophila larval axons, owing to decreased kinesin 1 attachment to microtubules and/or membranes, and reduced ATPase activity. Together, we propose that GSK3β phosphorylation fine-tunes kinesin 1 movement in vivo via differential phosphorylation, unraveling the complex in vivo regulatory mechanisms that exist during axonal motility of cargos attached to multiple kinesin 1 and dynein motors.

Lamin C is required to establish genome organization after mitosis

Background The dynamic 3D organization of the genome is central to gene regulation and development. The nuclear lamina influences genome organization through the tethering of lamina-associated domains (LADs) to the nuclear periphery. Evidence suggests that lamins A and C are the predominant lamins involved in the peripheral association of LADs, potentially serving different roles. Results Here, we examine chromosome architecture in mouse cells in which lamin A or lamin C are downregulated. We find that lamin C, and not lamin A, is required for the 3D organization of LADs and overall chromosome organization. Striking differences in localization are present as cells exit mitosis and persist through early G1 and are linked to differential phosphorylation. Whereas lamin A associates with the nascent nuclear envelope (NE) during telophase, lamin C remains in the interior, surrounding globular LAD aggregates enriched on euchromatic regions. Lamin C association with the NE is delayed until several hours into G1 and correlates temporally and spatially with the post-mitotic NE association of LADs. Post-mitotic LAD association with the NE, and global 3D genome organization, is perturbed only in cells depleted of lamin C, and not lamin A. Conclusions Lamin C regulates LAD dynamics during exit from mitosis and is a key regulator of genome organization in mammalian cells. This reveals an unexpectedly central role for lamin C in genome organization, including inter-chromosomal LAD-LAD segregation and LAD scaffolding at the NE, raising intriguing questions about the individual and overlapping roles of lamin A/C in cellular function and disease.

Comparative Proteomics and Phosphoproteomics Analysis Reveal the Possible Breed Difference in Yorkshire and Duroc Boar Spermatozoa

Sperm cells are of unique elongated structure and function, the development of which is tightly regulated by the existing proteins and the posttranslational modifications (PTM) of these proteins. Based on the phylogenetic relationships of various swine breeds, Yorkshire boar is believed to be distinctly different from Duroc boar. The comprehensive differential proteomics and phosphoproteomics profilings were performed on spermatozoa from both Yorkshire and Duroc boars. By both peptide and PTM peptide quantification followed by statistical analyses, 167 differentially expressed proteins were identified from 1,745 proteins, and 283 differentially expressed phosphopeptides corresponding to 102 unique differentially phosphorylated proteins were measured from 1,140 identified phosphopeptides derived from 363 phosphorylated proteins. The representative results were validated by Western blots. Pathway enrichment analyses revealed that majority of differential expression proteins and differential phosphorylation proteins were primarily concerned with spermatogenesis, male gamete generation, sperm motility, energy metabolism, cilium morphogenesis, axonemal dynein complex assembly, sperm–egg recognition, and capacitation. Remarkably, axonemal dynein complex assembly related proteins, such as SMCP, SUN5, ODF1, AKAP3, and AKAP4 that play a key regulatory role in the sperm physiological functions, were significantly higher in Duroc spermatozoa than that of Yorkshire. Furthermore, phosphorylation of sperm-specific proteins, such as CABYR, ROPN1, CALM1, PRKAR2A, and PRKAR1A, participates in regulation of the boar sperm motility mainly through the cAMP/PKA signal pathway in different breeds, demonstrating that protein phosphorylation may be an important mechanism underlying the sperm diversity. Protein–protein interaction analysis revealed that the 14 overlapped proteins between differential expression proteins and differential phosphorylation proteins potentially played a key role in sperm development and motility of the flagellum, including the proteins ODF1, SMCP, AKAP4, FSIP2, and SUN5. Taken together, these physiologically and functionally differentially expressed proteins (DEPs) and differentially expressed phosphorylated proteins (DPPs) may constitute the proteomic backgrounds between the two different boar breeds. The validation will be performed to delineate the roles of these PTM proteins as modulators of Yorkshire and Duroc boar spermatozoa.

Cell Cycle Regulates Phosphorylation of RUNX1 to Modulate Megakaryocyte-Erythroid Progenitor Fate Specification

Human Megakaryocytic-Erythroid Progenitors (MEP) produce megakaryocytic progenitors (MkP) and erythroid progenitors (ErP). Though some of the players have been identified, the molecular mechanisms underlying the MEP fate decision have not yet been determined. Using a functional single cell CFU-Mk/E assay, and single-cell RNA sequencing (scRNAseq), we have revealed that MEP cell cycling regulates MEP fate decisions with decreased cycling promoting Mk fate commitment and increased cycling promoting E fate commitment (Lu et al, Cell Reports, 2018). Our data point to RUNX1 (aka AML1), already known to be important for both Mk and E maturation, as playing a key role in MEP fate determination. RUNX1 target genes vary significantly between Common Myeloid Progenitors (CMP), MEP, MkP, and ErP. For example, the RUNX1 targets MPL, FLI1, and THBS1 are higher in MkP than in MEP and lowest in CMP and ErP. Analysis of scRNAseq data indicates that 11.8% and 9.3% of differentially (adj.p < 0.05, fold change > 2) expressed genes are predicted RUNX1 targets when comparing MEP to MkP and MEP to ErP, respectively (p=2.9e-8 and 5.6e-16). However, RUNX1 mRNA levels do not change significantly between CMP, MEP, MkP and ErP. We therefore assessed whether total RUNX1 protein levels and post-translational modifications change with Mk and E lineage commitment. Intracellular staining normalized to levels in CMP revealed that total RUNX1 protein is elevated 1.34-fold from CMP to MEP, is highest (1.65-fold) in MkP, and is intermediate in ErP (1.5-fold) consistent with RUNX1 perhaps promoting Mk over E fate. RUNX1 can be phosphorylated by cyclin dependent serine/threonine kinases (CDKs) as well as Src tyrosine kinases. Significant differences in RUNX1 phosphoprotein levels were revealed by intracellular FACS assays for differential phosphorylation of RUNX1. Phosphoserine modified RUNX1 (Ser21, Ser276, and Ser 397) levels, which likely reflect active RUNX1, are highest in MkP (1.28-fold over MEP), and diminished in ErP (-1.32-fold over MEP). To determine whether RUNX1 and phosphor-RUNX1 variants affect MEP commitment, we overexpressed wildtype (WT) RUNX1 or RUNX1-S4D (phospho-mimetic on S249, S277, T273, S276 residues). WT RUNX1 overexpression affected fate specification of primary MEP (p<0.05) with an increase in the Mk-only colony percentage from 24% to 43% at the expense of E-only colonies (from 26% to 16%) with little change in bipotent CFU-Mk/E. RUNX1-S4D more dramatically (p<0.04) increased Mk-only colonies (from 25% to 75%) and decreased E-only (from 25% to < 4%) and Mk/E (from 50% to 21%) colonies. Surprisingly, overexpression of RUNX1-S4D in FACSorted primary human ErP caused them to develop more (p<0.01) Mk/E-colonies (from 12% to 60%) with a dramatic decrease in E-only colonies (from 85% to 28%) suggesting that the E commitment of ErP is reversed (or reprogrammed) by activated RUNX1. In contrast, RUNX1 variants mimicking tyrosine phosphorylation (RUNX1-Y6D) promote E fate specification. Tyrosine phosphorylation on RUNX1 is mediated by Src family kinases (Huang te al, Genes Dev., 2012). We found that Src kinase inhibition results in increasing Mk lineage bias (from 20% to 71%) suggest differential phosphorylation of RUNX1 plays a pivotal role in MEP fate specification. Epigenetic modifications induced by phospho-serine vs phospho-tyrosine mimetics are currently under evaluation. Combining our data on cell cycle control of MEP with RUNX1 reveals that RUNX1 inhibition prevents the Mk bias induced by slowing the cell cycle. For example, the RUNX1 inhibitor Ro5-3335 causes MEP to have an Er bias from 27% to 61% while the mTOR inhibitor Rapamycin (slows MEP cell cycle) induces a Mk bias from 19% to 52%. The combination of Ro5-3335 plus Rapa results in increased Er from 27% to 45% suggest that RUNX1 influences MEP fate specification downstream of the cell cycle machinery. In summary, the phosphorylation status of RUNX1 in primary human hematopoietic progenitors regulates MEP fate commitment. Detailed mechanisms linking the cell cycle machinery to RUNX1 protein levels and function toward Mk vs E specification are being explored. Disclosures No relevant conflicts of interest to declare.

KRSA: Network-based Prediction of Differential Kinase Activity from Kinome Array Data

MotivationPhosphorylation by serine-threonine and tyrosine kinases is critical for determining protein function. Array-based approaches for measuring multiple kinases allow for the testing of differential phosphorylation between conditions for distinct sub-kinomes. While bioinformatics tools exist for processing and analyzing such kinome array data, current open-source tools lack the automated approach of upstream kinase prediction and network modeling. The presented tool, alongside other tools and methods designed for gene expression and protein-protein interaction network analyses, help the user better understand the complex regulation of gene and protein activities that forms biological systems and cellular signaling networks.ResultsWe present the Kinome Random Sampling Analyzer (KRSA), a web-application for kinome array analysis. While the underlying algorithm has been experimentally validated in previous publications, we tested the full KRSA application on dorsolateral prefrontal cortex (DLPFC) in male (n=3) and female (n=3) subjects to identify differential phosphorylation and upstream kinase activity. Kinase activity differences between males and females were compared to a previously published kinome dataset (11 female and 7 male subjects) which showed similar patterns to the global phosphorylation signal. Additionally, kinase hits were compared to gene expression databases for in silico validation at the transcript level and showed differential gene expression of kinases.Availability and implementationKRSA as a web-based application can be found at http://bpg-n.utoledo.edu:3838/CDRL/KRSA/. The code and data are available at https://github.com/kalganem/KRSA.Supplementary informationSupplementary data are available online.

The Differential Phosphorylation-Dependent Signaling and Glucose Immunometabolic Responses Induced during Infection by Salmonella Enteritidis and Salmonella Heidelberg in Chicken Macrophage-like cells

Salmonella is a burden to the poultry, health, and food safety industries, resulting in illnesses, food contamination, and recalls. Salmonella enterica subspecies enterica Enteritidis (S. Enteritidis) is one of the most prevalent serotypes isolated from poultry. Salmonella enterica subspecies enterica Heidelberg (S. Heidelberg), which is becoming as prevalent as S. Enteritidis, is one of the five most isolated serotypes. Although S. Enteritidis and S. Heidelberg are almost genetically identical, they both are capable of inducing different immune and metabolic responses in host cells to successfully establish an infection. Therefore, using the kinome peptide array, we demonstrated that S. Enteritidis and S. Heidelberg infections induced differential phosphorylation of peptides on Rho proteins, caspases, toll-like receptors, and other proteins involved in metabolic- and immune-related signaling of HD11 chicken macrophages. Metabolic flux assays measuring extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) demonstrated that S. Enteritidis at 30 min postinfection (p.i.) increased glucose metabolism, while S. Heidelberg at 30 min p.i. decreased glucose metabolism. S. Enteritidis is more invasive than S. Heidelberg. These results show different immunometabolic responses of HD11 macrophages to S. Enteritidis and S. Heidelberg infections.

The STRIPAK signaling complex regulates phosphorylation of GUL1, an RNA-binding protein that shuttles on endosomes

The striatin-interacting phosphatase and kinase (STRIPAK) multi-subunit signaling complex is highly conserved within eukaryotes. In fungi, STRIPAK controls multicellular development, morphogenesis, pathogenicity, and cell-cell recognition, while in humans, certain diseases are related to this signaling complex. To date, phosphorylation and dephosphorylation targets of STRIPAK are still widely unknown in microbial as well as animal systems. Here, we provide an extended global proteome and phosphoproteome study using the wild type as well as STRIPAK single and double deletion mutants from the filamentous fungus Sordaria macrospora. Notably, in the deletion mutants, we identified the differential phosphorylation of 129 proteins, of which 70 phosphorylation sites were previously unknown. Included in the list of STRIPAK targets are eight proteins with RNA recognition motifs (RRMs) including GUL1. Knockout mutants and complemented transformants clearly show that GUL1 affects hyphal growth and sexual development. To assess the role of GUL1 phosphorylation on fungal development, we constructed phospho-mimetic and -deficient mutants of GUL1 residues S180, S216, and S1343. While the S1343 mutants were indistinguishable from wildtype, phospho-deficiency of S180 and S216 resulted in a drastic reduction in hyphal growth and phospho-deficiency of S216 also affects sexual fertility. These results thus suggest that differential phosphorylation of GUL1 regulates developmental processes such as fruiting body maturation and hyphal morphogenesis. Moreover, genetic interaction studies provide strong evidence that GUL1 is not an integral subunit of STRIPAK. Finally, fluorescence microcopy revealed that GUL1 co-localizes with endosomal marker proteins and shuttles on endosomes. Here, we provide a new mechanistic model that explains how STRIPAK-dependent and - independent phosphorylation of GUL1 regulates sexual development and asexual growth.Author SummaryIn eukaryotes, the striatin-interacting phosphatase and kinase (STRIPAK) multi-subunit signaling complex controls a variety of developmental processes, and the lack of single STRIPAK subunits is associated with severe developmental defects and diseases. However, in humans, animals, as well as fungal microbes, the phosphorylation and dephosphorylation targets of STRIPAK are still largely unknown. The filamentous fungus Sordaria macrospora is a well-established model system used to study the function of STRIPAK, since a collection of STRIPAK mutants is experimentally accessible. We previously established an isobaric tag for relative and absolute quantification (iTRAQ)-based proteomic and phosphoproteomic analysis to identify targets of STRIPAK. Here, we investigate mutants that lack one or two STRIPAK subunits. Our analysis resulted in the identification of 129 putative phosphorylation targets of STRIPAK including GUL1, a homolog of the RNA-binding protein SSD1 from yeast. Using fluorescence microscopy, we demonstrate that GUL1 shuttles on endosomes. We also investigated deletion, phospho-mimetic, and -deletion mutants and revealed that GUL1 regulates sexual and asexual development in a phosphorylation-dependent manner. Collectively, our comprehensive genetic and cellular analysis provides new fundamental insights into the mechanism of how GUL1, as a STRIPAK target, controls multiple cellular functions.