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GRK2 influences the phosphorylation of PER proteins by LC-MS/MS

ABSTRACT: To test whether GRK2 influences the phosphorylation of PER proteins, we used mass spectrometry to identify residues in V5-tagged murine PER2 that may be differentially phosphorylated upon GRK2 overexpression in Neuro2A cells.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Cell Culture

SUBMITTER: kerwin chiang

LAB HEAD: Cheng-Kang Chiang

PROVIDER: PXD002290 | Pride | 2015-07-22

REPOSITORIES: Pride

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			Action	DRS
	Kerwin_20150310_in-gel_sample10_Run1.raw	Raw
	Kerwin_20150310_in-gel_sample11_Run1.raw	Raw
	Kerwin_20150310_in-gel_sample12_Run1.raw	Raw
	Kerwin_20150310_in-gel_sample13_Run1.raw	Raw
	Kerwin_20150310_in-gel_sample14_Run1.raw	Raw

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Project description:Voltage gated calcium channels play a central role in regulating the electrical and biochemical properties of neurons and muscle cells. Cells coordinate the expression of voltage gated calcium channels with the expression of other proteins that regulate membrane potential and calcium homeostasis. We report that the C-terminus of CaV 1.2, an L-type calcium channel (LTC) contains a C-terminal fragment that translocates to the nucleus and regulates transcription. This calcium channel associated transcription factor (CCAT) associates with transcriptional co-regulators such as p54nrb/NonO and binds to endogenous promoters. CCAT regulates the expression of gap junctions, sodium calcium exchangers, NMDA receptors, potassium channels and other proteins that regulate neuronal signaling. Electrical activity and developmental processes regulate the nuclear localization of CCAT, suggesting that the CCAT integrates information about the number of LTCs with information about the developmental history and electrical activity of a cell. These findings provide the first evidence that voltage gated calcium channels can directly activate transcription and suggest a novel mechanism linking voltage gated channels to the function and differentiation of excitable cells. Experiment Overall Design: The goal of these experiments was to identify the genes that are regulated by CCAT, a novel transcription factor derived from the C-terminus of CaV1.2. Neuro2A neuroblastoma cells were transfected with the last 503 AA of CaV1.2 which is full length CCAT (CCAT FL) or with the last 280 AA of CaV1.2, a form of CCAT that lacks the transcriptional activation domain (CCAT DTA). The mRNA from either CCAT FL or CCAT DTA expressing cells was hybridized to Agilent mouse genome microarrays along with mRNA from untransfected neuro2A cells (CCAT FL or DTA A series). Subsequent investigation revealed that transfection with the PA1 plasmid by itself increases the expression of some genes. To control for this effect we compared Neuro2A cells transfected with full length CCAT (in PA1) with Neuro2A cells transfectected with PA1 alone (CCAT FL B series). The microarray data was analyzed with the Rossetta Luminator gene expression data analysis system.

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GRK2 influences the phosphorylation of PER proteins by LC-MS/MS

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