Brain specific kinase-1 BRSK1/SAD-B associates with lipid rafts: modulation of kinase activity by lipid environment.
Brain specific kinases 1 and 2 (BRSK1/2, also named SAD kinases) are serine-threonine kinases specifically expressed in the brain, and activated by LKB1-mediated phosphorylation of a threonine residue at their T-loop (Thr189/174 in human BRSK1/2). BRSKs are crucial for establishing neuronal polarity, and BRSK1 has also been shown to regulate neurotransmitter release presynaptically. How BRSK1 exerts this latter function is unknown, since its substrates at the synaptic terminal and the mechanisms modulating its activity remain to be described. Key regulators of neurotransmitter release, such as SNARE complex proteins, are located at membrane rafts. Therefore we initially undertook this work to check whether BRSK1 also locates at these membrane microdomains. Here we show that brain BRSK1, but not BRSK2, is palmitoylated, and provide biochemical and pharmacological evidences demonstrating that a pool of BRSK1, but not BRSK2 or LKB1, localizes at membrane lipid rafts. We also show that raft-associated BRSK1 has higher activity than BRSK1 from non-raft environment, based on a higher T-loop phosphorylation at Thr-189. Further, recombinant BRSK1 activity increased 3-fold when assayed with small multilamellar vesicles (SMV) generated with lipids extracted from synaptosomal raft fractions. A similar BRSK1-activating effect was obtained with synthetic SMV made with phosphatidylcholine, cholesterol and sphingomyelin, mixed in the same molar ratio at which these three major lipids are present in rafts. Importantly, SMV also enhanced the activity of a constitutively active BRSK1 (T189E), underpinning that interaction with lipid rafts represents a new mechanism of BRSK1 activity modulation, additional to T-loop phosphorylation.
Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, Barcelona, Spain.
Arantza
A
Rodríguez-Asiain
Gerard
G
Ruiz-Babot
Walter
W
Romero
Roger
R
Cubí
Tatiana
T
Erazo
Ricardo M
RM
Biondi
Jose R
JR
Bayascas
Jose
J
Aguilera
Nestor
N
Gómez
Carles
C
Gil
Enrique
E
Claro
Jose M
JM
Lizcano
D000091162:AMP-Activated Protein Kinase Kinases; D000818:Animals; D016367:Baculoviridae; D001921:Brain / Q000502:physiology*; D004926:Escherichia coli; D005333:Fetus; D057809:HEK293 Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D054878:Lipoylation; D021962:Membrane Microdomains / Q000378:metabolism*; D008567:Membranes, Artificial; D051379:Mice; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D017433:Protein Structure, Secondary; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*; D009435:Synaptic Transmission / Q000502:physiology*; D013574:Synaptosomes / Q000378:metabolism*; D013912:Threonine / Q000378:metabolism
D047908:Intracellular Signaling Peptides and Proteins; D008567:Membranes, Artificial; D011994:Recombinant Proteins; D013912:Threonine; C523084:BRSK1 protein, human; C507732:Brsk2 protein, human; D017346:Protein Serine-Threonine Kinases; C488755:Stk11 protein, rat; D000091162:AMP-Activated Protein Kinase Kinases