Abstract
Brain-specific kinases 1 and 2 (BRSK1/2) are AMP-activated protein kinase (AMPK)-related kinases that are highly expressed in mammalian forebrain. Studies using transgenic animal models have implicated a role for these kinases in the establishment of neuronal polarity. BRSK1 and BRSK2 are activated by phosphorylation of a threonine residue in the T-loop activation segment of the kinase domain. In vitro studies have demonstrated that LKB1, an upstream kinase in the AMPK cascade, can catalyze this phosphorylation. However, to date, a detailed comparative analysis of the molecular regulation of BRSK1/2 has not been undertaken. Here we present evidence that excludes another upstream kinase in the AMPK cascade, Ca(2+)/calmodulin-dependent protein kinase kinase beta, from a role in activating BRSK1/2. We show that equivalent mutations in the ubiquitin-associated domains of the BRSK isoforms produce differential effects on the activation of BRSK1 and BRSK2. Contrary to previous reports, activation of cAMP-dependent protein kinase does not affect BRSK1 or BRSK2 activity in mammalian cells. Furthermore, stimuli that activate AMPK had no effect on BRSK1/2. Finally, we provide evidence suggesting that protein phosphatase 2C is a likely candidate for catalyzing the dephosphorylation and inactivation of BRSK1/2.
Highlights
Previous studies have demonstrated that LKB1 is phosphorylated by cAMP-dependent protein kinase (PKA) at serine 431 in the mouse protein, phosphorylation at this residue had no detectable effect on LKB1 activity [11, 12]
We show that an equivalent mutation within the ubiquitin-associated (UBA) domains of BRSK1 and BRSK2 has differential effects on their activity
We found that PKA does not directly regulate the activity, or activation by LKB1, of BRSK1 or BRSK2
Summary
Plasmids—BRSK2 was cloned into the pcDNA3.1a/Myc-His mammalian expression vector (Invitrogen). pCMV-BRSK1-HA and cDNA encoding human LKB1 were kind gifts from Prof. CCL13 cells were transfected with BRSK1 (HA-tagged), wild-type (WT) BRSK2, or BRSK2 harboring the T174A mutation (both Myc-tagged) and infected with varying amounts of adenovirus, measured as pfu/cell, containing either LKB1 (A and B) or CaMKK (FLAG-tagged (C)). BRSK1 and BRSK2 were immunoprecipitated from cell lysates (100 g) with either anti-HA or anti-Myc antibodies, and kinase activity was assayed using the synthetic peptide substrate LNR (A and B). Protein expression was analyzed by Western blotting of cell lysates (30 g) with anti-HA (BRSK1), anti-Myc (BRSK2), anti-LKB1, or anti-FLAG (CaMKK) antibodies, and in each case, a representative blot is shown. Expression of LKB1 had no obvious effect on the level of expression of either BRSK1 or BRSK2, as AMPK activity was measured by phosphorylation of the SAMS judged by Western blotting. No activation of this mutant form of BRSK2 was considered significant at p Ͻ 0.05
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