Differential effects of PKA-controlled CaMKK2 variants on neuronal differentiation

Abstract

Regulation between protein kinases is critical for the establishment of signaling pathways/networks to 'orchestrate' cellular processes. Besides posttranslational phosphorylation, alternative pre-mRNA splicing is another way to control kinase properties, but splicing regulation between two kinases and the effect of resulting variants on cells has barely been explored. Here we examined the effect of the protein kinase A (PKA) pathway on the alternative splicing and variant properties of the Ca²⁺/calmodulin-dependent protein kinase kinase 2 (CaMKK2) gene in B35 neuroblastoma cells. Inclusion of the exon 16 of CaMKK2 was significantly reduced by H89, a PKA selective inhibitor. Consistently, overexpressed PKA strongly promoted the exon inclusion in a CaMKK2 sequence-dependent way in splicing reporter assays. In vitro, purified CaMKKβ1 variant proteins were found to be kinase-active. In cells, they were differentially phosphorylated by PKA. In RNA interference assays, CaMKKβ1 was found to be essential for forskolin-induced neurite growth. Interestingly, overexpression of the variant without exon 16 (-E16) promoted neurite elongation while the other one (+E16) promoted neurite branching; in contrast, reduction of the latter one enhanced neurite elongation. Moreover, the variants are differentially expressed and the exon 16-containing transcripts highly enriched in the brain, particularly the cerebellum and hippocampus. Thus, PKA regulates the alternative splicing of CaMKK2 to produce variants that differentially modulate neuronal differentiation. Taken together with the many distinct variants of kinases, alternative splicing regulation likely adds another layer of modulation between protein kinases in cellular signaling networks.

Convergent lines of evidence support CAMKK2 as a schizophrenia susceptibility gene

“…One study also showed the enzyme encoded by CAMKK2 can phosphorylate AMP-activated protein kinase. 68 The CAMKK2 gene has the strongest expression in brain 31 and it influences signaling cascades that are involved with learning and memory, 32,69 neuronal differentiation and migration, 70 neurite outgrowth 34,35,70 and synapse formation.…”

Calcium/Calmodulin-dependent Protein Kinase Kinase 2: Roles in Signaling and Pathophysiology

“…Two major transcripts are generated by use of polyadenylation sites present in the last two exons. Additionally, CaMKK2 transcripts can be generated by alternative splicing of internal exons 14 and/or 16; this mechanism produces variants with different roles in neuronal differentiation (16,17). PKA and CaMKIV have been reported to be involved in regulation of alternative splicing of the CaMKK2 transcript (17).…”

“…CaMKK and CaMKI co-localize with ␤PIX (p21-activated kinase-interacting exchange factor) and GIT1 (G-protein-coupled receptor kinase-interacting protein 1) in dendritic spines as part of a multiprotein complex that regulates actin dynamics (43). Differential splicing and phosphorylation of critical Ser residues affect the ability of CaMKK2 to control dendrite/axon formation (17,27). Thus, a CaMKK2/CaMKI cascade regulates learning-induced neuronal cytoskeleton remodeling associated with memory formation.…”

“…CaMKK2 may regulate the stability and/or endosome recycling of PYK2, thus tuning the cross-talk between integrin signaling and the TLR4-dependent cascade. Thus, in macrophages as well as in neurons, CaMKK2 participates in signaling pathways controlling cytoskeleton remodeling and morphological changes induced by external stimuli (17,27,37).…”

“…2). Generation and/or splicing of the primary transcript is a regulated event, and in prostate cancer cells, transcription can be regulated by androgens (16,17,79,80). CaMKK2 protein is also subject to numerous post-translational modifications ( Fig.…”