Investigating the regulation of brain-specific kinases 1 and 2 by phosphorylation

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.

FIGURE 1.

BRSK expression and activity. Mouse tissue homogenates (30 μg) were analyzed by Western blotting using anti-BRSK1 or anti-BRSK2 antibodies (A). Protein loading was visualized by staining the membranes with Coomassie Brilliant Blue (CBB) and is shown below each blot. BRSK1/2 were immunoprecipitated from mouse tissue homogenates (100 μg) with anti-BRSK1 or anti-BRSK2 antibodies, and kinase activity of the immune complexes was measured using the peptide substrate LNR (B). Results are plotted as pmol/min/mg of lysate and are the mean of at least three experiments ± S.D.

FIGURE 2.

Activation of BRSK1 and BRSK2 by LKB1 but not CaMKKβ. 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 CaMKKβ (FLAG-tagged (C)). BRSK1 and BRSK2 were immunoprecipitated from cell lysates (100 μg) with either anti-HA or anti-Myc antibodies, and kinase activity was assayed using the synthetic peptide substrate LNR (A and B). Endogenous AMPK was immunoprecipitated from cell lysates, and activity was determined by the SAMS peptide assay (C). Results are plotted as pmol/min/mg of lysate and are the mean of at least three experiments ± S.D.; *, p < 0.05 or **, p < 0.01, significantly different from activity in the absence of LKB1. Protein expression was analyzed by Western blotting of cell lysates (30 μg) with anti-HA (BRSK1), anti-Myc (BRSK2), anti-LKB1, or anti-FLAG (CaMKKβ) antibodies, and in each case, a representative blot is shown.

FIGURE 3.

Activation of BRSK1 and BRSK2 mutants by LKB1. CCL13 cells were transfected with wild-type BRSK2 (WT) or BRSK2 harboring either the T174A or the T174E mutation (A), wild-type BRSK1 or BRSK1 harboring the G353A mutation (B), or wild-type BRSK2 or BRSK2 harboring the G310A mutation (C). Cells were infected with the indicated amount of adenovirus containing LKB1 (10 pfu/cell in A), and BRSK1 and BRSK2 activity was measured in immune complexes using the LNR peptide assay. Results are plotted either as the percentage of activity of wild-type BRSK2 in the presence of LKB1 (A) or pmol/min/mg of lysate (B and C) and are the mean of at least three experiments ± S.D.; *, p < 0.05 or **, p < 0.01, significantly different from activity in the absence of LKB1. In each case, a representative Western blot is included to show protein expression levels. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

FIGURE 4.

Activation of PKA by forskolin has no effect on BRSK1 or BRSK2 activity. CCL13 (A), HEK293 (B), or SH-SY5Y (C) cells were transfected with BRSK1, wild-type (WT) BRSK2, or BRSK2 harboring the T174A mutation. Additionally, CCL13 cells were infected with adenovirus containing LKB1 (10 pfu/cell) as indicated on the figure. 48 h after transfection, cells were incubated with 20 μm forskolin or a vehicle control for 15 min prior to harvesting. BRSK1 and BRSK2 activity was determined in immune complexes by the LNR peptide assay. Results are plotted as pmol/min/mg of lysate and are the mean of at least three experiments ± S.D. Activation of PKA was monitored by determining the level of phosphorylation of CREB relative to total CREB by Western blotting. Bar charts showing the ratio of phospho-CREB (pCREB):total CREB are shown (±S.D.) as well as representative blots. Significant differences in the ratio of phospho-CREB in the presence of forskolin relative to vehicle are shown, *, p < 0.05.

FIGURE 5.

Activation of BRSK1 and BRSK2 by LKB1 mutants. CCL13 cells were co-transfected with BRSK1 (A) or BRSK2 (B) and either wild-type (WT) LKB1 or the various LKB1 mutants, as indicated. BRSK activity was determined using the LNR peptide assay. Results are plotted as pmol/min/mg of lysate and are the mean of at least three experiments ± S.D. In each case, representative Western blots show protein expression levels. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. C, 48 h after transfection with BRSK2 and the various LKB1 mutants indicated, CCL13 cells were treated with either 20 μm forskolin or vehicle control for 15 min prior to harvesting and determination of BRSK2 activity, as above. Results are plotted as pmol/min/mg of lysate and are the mean of three experiments ± S.D.

FIGURE 6.

Effect of various treatments on BRSK2 activity. SH-SY5Y cells were incubated with ionomycin (1 μm, 5 min), carbachol (10 μm, 5 min), or sorbitol (0.5 m, 30 min). BRSK2 was immunoprecipitated from SH-SY5Y lysates (100 μg) and activity measured by LNR substrate assay (A). SH-SY5Y cells were treated with okadaic acid (200 nm, 45 min), and BRSK2 activity was determined as above (B). In both cases, results are plotted as pmol/min/mg and are the average of at least three experiments ± S.D. Phosphatase inhibition by okadaic acid was monitored by determining the levels of phospho-CREB (pCREB) relative to total CREB (B, bottom panel). C, active BRSK2 immunoprecipitated from CCL13 co-expressing LKB1 was incubated with increasing amounts of recombinant PP2Cα for 20 min. Following washing of the immune complexes to remove PP2C, BRSK2 activity was determined. Results are plotted as the percentage of activity determined in the absence of PP2C and are the mean value from three experiments ± S.D. Western blot analysis was used to verify the presence of equal amounts of BRSK2 (C, bottom panel).