9421 ser338 phospho craf ser338  (Cell Signaling Technology Inc)

Journal: The Journal of Biological Chemistry

Article Title: 14-3-3 interaction with phosphodiesterase 8A sustains PKA signaling and downregulates the MAPK pathway

doi: 10.1016/j.jbc.2024.105725

Figure Lengend Snippet: Proposed model for the role of 14-3-3ζ in regulation of PDE8A activity and downstream signaling. 1, binding of ligand to the membrane bound G protein–coupled receptor (GPCR) activates adenylate cyclase (AC). Activated AC converts ATP to cAMP. 2, cAMP thereby binds to the regulatory subunit (R1α) of PKA and facilitates dissociation of the catalytic subunit (PKA cat ). 3, PKA cat in turn phosphorylates CREB, PDE8A, and CRAF at Ser133, Ser359, and Ser259 residues, respectively. Phosphorylated CREB moves to the nucleus and promotes transcription of target genes. On the other hand, activated PDE8A binds to R1α and begins hydrolyzing cAMP by substrate channeling, thereby turning down the PKA signal. In addition, phosphorylation of CRAF facilitates 14-3-3ζ binding and inactivates MAPK pathway. 4, binding of 14-3-3ζ to the phosphorylated Ser359 residue of PDE8A, inhibits its phosphodiesterase activity and sustains the cAMP/PKA/CREB cascade. 5, in subcellular microdomains, 14-3-3ζ enhances the cAMP pool by inhibiting PDE8A activity associated with CRAF and promote inhibitory Ser259 phosphorylation of CRAF by PKA cat , thus downregulating of MEK/ERK pathway. The model was created using BioRender. ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; PDE8A, phosphodiesterase 8A.

Article Snippet: The membrane was blocked with 5% nonfat milk for 1 h followed by overnight incubation by the following primary antibodies, α-FLAG (Sigma, F7425), α-HA (Sigma, H3663), α-MYC (Merck, #05-724), α-14-3-3 pan (CST, #8312), α-14-3-3ζ (CST, #9639), α-GAPDH (BioBharati LifeScience, BB-AB0060), α-p-ERK1/2 (CST, #4370), total ERK1/2 (CST, #4696), α-pCREB (CST, #9198), total CREB (CST, #9197), α-p-Ser259 CRAF (CST, #9421), α-CRAF (BD Biosciences, 610152), α-GFP (Roche, 11814460001), α-HIS (Sigma, H1029), and α-PGK1 (Invitrogen, 459250).

Techniques: Activity Assay, Binding Assay, Membrane, Phospho-proteomics, Residue