Conage-Pough, J. E. | bioRxiv Science

bioRxiv2023

Differential substrate specificity of ERK, JNK, and p38 MAP kinases toward Connexin 43

Phosphorylation of connexin 43 (Cx43) is an important regulatory mechanism of gap junction (GJ) function. Cx43 is modified by several kinases on over 15 sites within its [~]140 amino acid-long C-terminus (CT). Phosphorylation of Cx43CT on S255, S262, S279, and S282 by ERK has been widely documented in several cell lines, by many investigators. Phosphorylation of these sites by JNK and p38, on the other hand, is not well-established. Indeed, ERK is a kinase activated by growth factors and is upregulated in diseases, such as cancer. JNK and p38, however, have a largely tumor-suppressive function due to their stress-activated and apoptotic role. We investigated substrate specificity of all three MAPKs toward Cx43CT, first by using purified proteins, and then in two cell lines (MDCK - non-cancerous, epithelial cells and porcine PAECs - pulmonary artery endothelial cells). Cx43 phosphorylation was monitored through gel-shift assays on an SDS-PAGE, immunodetection with phospho-Cx43 antibodies, and LC-MS/MS phosphoproteomic analyses. Our results demonstrate that p38 and JNK specificity differ from each other and from ERK. JNK has a strong preference for S255, S262, and S279, while p38 readily phosphorylates S262, S279, and S282. While we confirmed that ERK can phosphorylate all four serines (255, 262, 279, and 282), we also identified T290 as a novel ERK phosphorylation site. In addition, we assessed Cx43 GJ function upon activation or inhibition of each MAPK in PAECs. This work underscores the importance of delineating the effects of ERK, JNK, and p38 signaling on Cx43 and GJ function.

biochemistry↗

bioRxiv2021

Quantitative consequences of protein carriers in immunopeptidomics and tyrosine phosphorylation MS2 analyses

Utilizing a protein carrier in combination with isobaric labeling to "boost" the signal of other low-level samples in multiplexed analyses has emerged as an attractive strategy to enhance data quantity while minimizing protein input in mass spectrometry analyses. Recent applications of this approach include pMHC profiling and tyrosine phosphoproteomics, two applications that are often limited by large sample requirements. While including a protein carrier has been shown to increase the number of identifiable peptides in both applications, the impact of a protein carrier on quantitative accuracy remains to be thoroughly explored, particularly in relevant biological contexts where samples exhibit dynamic changes in abundance across peptides. Here, we describe two sets of analyses comparing MS2-based quantitation using a 20x protein carrier in pMHC analyses and a high ([~]100x) and low ([~]9x) protein carrier in pTyr analyses, using CDK4/6 inhibitors and EGF stimulation to drive dynamic changes in the immunopeptidome and phosphoproteome, respectively. In both applications, inclusion of a protein carrier resulted in an increased number of MHC peptide or phosphopeptide identifications, as expected. At the same time, quantitative accuracy was adversely affected by the presence of the protein carrier, altering interpretation of the underlying biological response to perturbation. Moreover, for tyrosine phosphoproteomics, the presence of high levels of protein carrier led to a large number of missing values for endogenous phosphopeptides, leading to fewer quantifiable peptides relative to the "no-boost" condition. These data highlight the unique limitations and future experimental considerations for both analysis types and provide a framework for assessing quantitative accuracy in protein carrier experiments moving forward.

bioengineering↗