PXD031262

PXD031262 is an original dataset announced via ProteomeXchange.

Title	Identification of phosphorylation and other post-translational modifications in the central C4-C5 domains of murine cardiac myosin binding protein C
Description	Background: Cardiac myosin binding protein C (cMyBPC) is a critical multi-domain protein that modulates myosin cross bridge behavior and cardiac contractility. cMyBPC is principally regulated by phosphorylation of the residues within the M-domain of its N-terminus, which is essential for proper cardiac function. However, not much is known about the phosphorylation or additional post-translational modification (PTM) landscape of other regions of the molecule, including the enigmatic central C4C5 domains. Methods and Results: To this end, we utilized in vitro kinase assays and mass spectrometry to identify kinase-specific phosphorylation sites within the murine cMyBPC C4C5 domains. The presence of phosphorylation outside the M-domain was confirmed in vivo using mouse models expressing cMyBPC with non-phosphorylatable serine to alanine substitutions. Purified recombinant mouse C4C5 domain constructs were incubated with 13 different kinases, and the six strongest kinases were chosen for mass spectrometry analysis (ribosomal protein S6 kinase A3, RSK2; cAMP dependent protein kinase, PKA; protein kinase cGMP-dependent 1, PKG1; AMP-activated protein kinase, AMPK; protein kinase D2, PKD2; and casein kinase 2, CK2). A total of 26 unique phosphorylated peptides were found, representing 13 different phosphorylation sites including 10 novel sites that had not been previously described. Through parallel reaction monitoring experiments, serine (S) sites S690, S589, S546, and S730, targeted by PKA and PKG1 were determined to be the most strongly phosphorylated. Mutagenesis and subsequent kinase experiments showed that S690 is the most significant site of PKA-induced phosphorylation, while both S690 and S589 were sites of PKG-induced phosphorylation, in vitro. In addition, we report 9 acetylation and 5 ubiquitination sites not previously found in the literature. Conclusions: Our findings showed evidence of additional novel phosphorylation, acetylation, and ubiquitination sites in the C4C5 domains of mouse cMyBPC. Although the precise function and regulation of the C4C5 domains of cMyBPC is not yet known, these PTMs demonstrate the possibility of additional layers of regulation and potential importance of the central domains of cMyBPC in cardiac health and disease.
HostingRepository	PRIDE
AnnounceDate	2022-05-04
AnnouncementXML	Submission_2022-05-04_14:44:58.186.xml
DigitalObjectIdentifier	https://dx.doi.org/10.6019/PXD031262
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Supported dataset by repository
PrimarySubmitter	Ling Li
SpeciesList	scientific name: Mus musculus (Mouse); NCBI TaxID: 10090;
ModificationList	ubiquitination signature dipeptidyl lysine; phosphorylated residue; acetylated residue; monohydroxylated residue
Instrument	Orbitrap Fusion Lumos

Revision	Datetime	Status	ChangeLog Entry
0	2022-01-26 06:30:25	ID requested
⏵ 1	2022-05-04 14:44:58	announced

Dataset with its publication pending

submitter keyword: cardiac myosin binding protein C, post translational modification, mass spectrometry

Julian E. Stelzer
contact affiliation	Department of Physiology and Biophysics, School of Medicine, Case Western Reserve University, Cleveland, OH 44106 USA
contact email	julian.stelzer@case.edu
lab head
Ling Li
contact affiliation	Cleveland Clinic
contact email	lil5@ccf.org
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/2022/05/PXD031262

PRIDE project URI

• PRIDE
  1. PXD031262
     1. Label: PRIDE project
     2. Name: Identification of phosphorylation and other post-translational modifications in the central C4-C5 domains of murine cardiac myosin binding protein C