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Proteomic analysis of affinity-purified praja2 complexes

ABSTRACT: Praja2 is a RING E3 ubiquitin ligase, widely expressed in mammalian cells and tissues which is involved in essential aspects of cell physiology. Several signaling pathways are regulated by praja2. The stability of regulatory PKA subunits is directly regulated by Praja2, which binds and tethers cAMP-dependent protein kinase (PKA) to intracellular membranes and organelles, ensuring efficient integration, propagation and amplification of the locally-generated cAMP to distinct target compartments. By regulating cAMP signaling, praja2 efficiently couples phosphorylation to ubiquitination of protein kinases, scaffolds and effectors, with important implication for neuronal activity, development, inflammatory responses, ciliogenesis, cell growth and metabolism. Dysregulation of praja2-regulated signaling pathways has been causally linked to the growth and progression of glioblastoma.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Kidney

DISEASE(S): Glioblastoma

SUBMITTER: CHIARA D'AMBROSIO

LAB HEAD: Andrea Scaloni

PROVIDER: PXD033734 | Pride | 2026-06-25

REPOSITORIES: Pride

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Project description:The second messenger cAMP acts via protein kinase A (PKA) to induce apoptosis by mechanisms that are poorly understood. Here, we assessed a role for mitochondria and analyzed gene expression in cAMP/PKA-promoted apoptosis by comparing wild-type (WT) S49 lymphoma cells and the S49 variant, D- (cAMP-deathless), which lacks cAMP-promoted apoptosis but has wild-type levels of PKA activity and cAMP-promoted G1 growth arrest. Treatment of WT, but not D-, S49 cells with 8-CPT-cAMP for 24 h induced loss of mitochondrial membrane potential, mitochondrial release of cytochrome c and Smac and increase in caspase-3 activity. Gene expression analysis (using Affymetrix 430 2.0 Arrays) revealed that WT and D- cells incubated with 8-CPT-cAMP have similar, but non-identical, extents of cAMP-regulated gene expression at 2h (~800 transcripts) and 6h (~1000 transcripts) (|Fold|>2, P<0.06); by contrast, at 24h ~2500 and ~1100 transcripts were changed in WT and D- cells, respectively. Using an approach that combined regression analysis, clustering and functional annotation to identify transcripts that showed differential expression between WT and D- cells, we found differences in cAMP-mediated regulation of mRNAs involved in transcriptional repression, apoptosis, the cell cycle, RNA splicing, Golgi and lysosomes. The 2 cell lines differed in CREB phosphorylation and expression of the transcriptional inhibitor Icer and in cAMP-regulated expression of genes in the Inhibitor of apoptosis (IAP) and Bcl families. The findings indicate that cAMP/PKA-promoted apoptosis of lymphoid cells occurs via mitochondrial-mediated events and imply that such apoptosis involves gene networks in multiple biochemical pathways. Experiment Overall Design: S49 D- cells were treated with 8-CPT-cAMP over the course of 24 hours. Cells were prepared for hybridization to microarrays at times 0-untreated, 2h, 6h, and 24h after treatment with 8-CPT-cAMP. Experimental replicates were as follows n=4 for time 0 and n=3 for 2, 6, and 24h post treatment.

			Action	DRS
	BAT_FLAG_PRAJA2.msf	Msf
	BAT_FLAG_PRAJA2.pdResult	Other
	BAT_FLAG_PRAJA2_1.raw	Raw
	BAT_FLAG_PRAJA2_10.raw	Raw
	BAT_FLAG_PRAJA2_11.raw	Raw

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Proteomic analysis of affinity-purified praja2 complexes

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