Phosphorylation of apolipoprotein-E at an atypical protein kinase CK2 PSD/E site in vitro.
Apolipoprotein-E (apoE) plays an important role in neuronal lipid transport and is thought to stabilize microtubules by preventing tau hyperphosphorylation. ApoE is also associated with insoluble amyloid detected in Alzheimer disease brain lesions. The apoE C-terminal shares several physicochemical features with alpha-synuclein, another neuronal apolipoprotein-like protein. Alpha-synuclein is phosphorylated by protein kinase CK2 (CK2) at an atypical PSD/E motif in vivo and in vitro. We identified a similar PSD/E motif in apoE and therefore investigated its potential phosphorylation by CK2 in vitro. When a [(32)P]-labeling approach was used, CK2 readily phosphorylated purified human apoE as well as recombinant forms of human apoE3 and apoE4. Using liquid chromatography mass spectrometry techniques, we mapped the major apoE CK2 phosphorylation site to Ser296 within the apoE PSD/E motif. We also found that apoE potently activated CK2 as demonstrated by increased CK2beta subunit autophosphorylation and by increased phosphorylation of tau when the latter was added to the kinase reaction mixtures. Other proteins such as apolipoprotein A-I and albumin did not effectively activate CK2. The phosphorylation of apoE by CK2 as well as the activation of CK2 by apoE may be relevant in vivo where apoE, CK2, and tau are co-localized with additional CK2 targets on neuronal microtubules.
Raftery
Mark
M
Bioanalytical Mass Spectrometry Facility, University of New South Wales, Sydney NSW 2052, Australia.
Campbell
Ross
R
Glaros
Elias N
EN
Rye
Kerry-Anne
KA
Halliday
Glenda M
GM
Jessup
Wendy
W
Garner
Brett
B
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D053318:Apolipoprotein E3; D053327:Apolipoprotein E4; D001057:Apolipoproteins E / Q000378:metabolism*; D047390:Casein Kinase II / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D008870:Microtubules / Q000378:metabolism; D008969:Molecular Sequence Data; D009474:Neurons / Q000378:metabolism; D010761:Phosphorus Radioisotopes / Q000378:metabolism; D010766:Phosphorylation; D020033:Protein Isoforms / Q000378:metabolism; D011817:Rabbits; D051381:Rats; D016875:tau Proteins / Q000378:metabolism
D053318:Apolipoprotein E3; D053327:Apolipoprotein E4; D001057:Apolipoproteins E; D008869:Microtubule-Associated Proteins; D010761:Phosphorus Radioisotopes; D020033:Protein Isoforms; D016875:tau Proteins; D047390:Casein Kinase II