PXD014985-2

PXD014985 is an original dataset announced via ProteomeXchange.

Title	CRISPR-Cas9/phosphoproteomics identifies Myosin Light Chain Kinase dependent signaling in kidney epithelial cells
Description	Prior studies have implicated myosin light chain kinase (MLCK) in the regulation of aqua-porin-2 (AQP2) in the renal collecting duct. To discover signaling targets of MLCK, we deleted the Mylk- gene to obtain MLCK-null mpkCCD cells and carried out comprehensive phosphopro-teomics using the SILAC method for quantification. Only 107 phosphopeptides were changed in abundance by MLCK deletion out of 1743 quantified over multiple replicates (29 decreased and 78 increased). One of the decreased phosphopeptides corresponded to the canonical target site in myosin regulatory light chain (MLC). Network analysis indicated that targeted phosphopro-teins clustered into distinct structural/functional groups: “acto-myosin,” “signaling,” “nuclear envelope,” “gene transcription,” “mRNA processing,” “energy metabolism,” “intermediate fila-ments,” “adherens junctions” and “tight junctions.” There was significant overlap between the derived MLCK signaling network and a previously determined PKA signaling network. Phalloidin labeling revealed that MLCK deletion inhibited the normal effect of vasopressin to depolymer-ize F-actin. Immunocytochemistry and electron microscopy demonstrated a defect in pro-cessing of early endosomes to late endosomes. In contrast, surface biotinylation studies showed no effect of MLCK deletion on AQP2 endocytosis. We conclude that MLCK is part of a multi-component signaling pathway that includes much more than simple regulation of conven-tional non-muscle myosins through MLC phosphorylation. Furthermore, vasopressin signaling in collecting duct cells involves both MLCK-dependent signaling and PKA-dependent signaling, which display significant overlap. Mapping MLCK targets revealed potential roles in both nu-cleus and cytoplasm. The latter includes proteins that regulate both actin polymerization and AQP2-endosomal processing from early to late endosomes.
HostingRepository	PRIDE
AnnounceDate	2024-10-22
AnnouncementXML	Submission_2024-10-22_04:57:58.304.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	CHIN-RANG YANG
SpeciesList	scientific name: Mus musculus (Mouse); NCBI TaxID: 10090;
ModificationList	phosphorylated residue; monohydroxylated residue; iodoacetamide derivatized residue; deamidated residue
Instrument	LTQ Orbitrap Elite

Revision	Datetime	Status	ChangeLog Entry
0	2019-08-12 02:21:43	ID requested
1	2020-03-04 00:08:34	announced
⏵ 2	2024-10-22 04:58:06	announced	2024-10-22: Updated project metadata.

Isobe K, Raghuram V, Krishnan L, Chou CL, Yang CR, Knepper MA, CRISPR-Cas9/phosphoproteomics identifies multiple noncanonical targets of myosin light chain kinase. Am J Physiol Renal Physiol, 318(3):F600-F616(2020) [pubmed]

10.1152/ajprenal.00431.2019;

submitter keyword: nuclear membrane, mass spectrometry, vasopressin,AQP2, endosomal trafficking, STED microscopy

Mark A Knepper
contact affiliation	Epithelial Systems Biology Laboratory Systems Biology Center Division of Intramural Research NHLBI, NIH USA
contact email	knepperm@nhlbi.nih.gov
lab head
CHIN-RANG YANG
contact affiliation	National Institutes of Health, USA
contact email	chin-rang.yang@nih.gov
dataset submitter

Dataset FTP location NOTE: Most web browsers have now discontinued native support for FTP access within the browser window. But you can usually install another FTP app (we recommend FileZilla) and configure your browser to launch the external application when you click on this FTP link. Or otherwise, launch an app that supports FTP (like FileZilla) and use this address: ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/03/PXD014985

PRIDE project URI

• PRIDE
  1. PXD014985
     1. Label: PRIDE project
     2. Name: CRISPR-Cas9/phosphoproteomics identifies Myosin Light Chain Kinase dependent signaling in kidney epithelial cells