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. 2022 Apr 10;298(5):101928. doi: 10.1016/j.jbc.2022.101928

Figure 1.

Figure 1

Phosphorylation of AMPKα1-T172 and SIK3 other than LKB1 and MST3. Lysates were prepared from HEK293T cells or mouse brains. They were fractionated on an SP HP column followed by NaCl gradient elution. Fractions were collected, and aliquots of 0.5 ml of each fraction were dialyzed against buffer A, followed by in vitro SIK3 and AMPK phosphorylation assays. A, fractions from WT HEK cells. AMPK phosphorylating activities were observed in more fractions than SIK3 phosphorylating activities. Both activities were present in more fractions than those containing LKB1 and MST3. For example, fractions 11 and 12 (300–330 mM NaCl) contained strong SIK3 and AMPK phosphorylation activities but no detectable LKB1 or MST3. Fraction 22 (1000 mM NaCl) had strong AMPK phosphorylation activity but no LKB1 or MST3. B, fractions from Lkb1 KO HEK cells. In the absence of LKB1, activities in fractions 6 and 7 (150–180 mM NaCl) and fractions 11 and 12 (300–330 mM NaCl) were still present. MST3 was present in fractions 5 and 6 (120–150 mM NaCl) but not fractions 10 to 13 (270–360 mM NaCl). C, fractions from Mst3 KO HEK cells. Phosphorylating activities in fractions 7 and 8 (180–210 mM NaCl) were reduced by not eliminating in Mst3 KO HEK cells. Phosphorylating activities existed in fractions 10 to 12 (270–330 mM NaCl). D, fractions from WT mouse brains. Fractions 12, 13, and 14 (330–390 mM NaCl) contained AMPK and SIK3 phosphorylating activities but neither LKB1 nor MST3. E, fractions from Mst3 KO mouse brains. Mst3 KO mice were generated. Their brains were isolated, and extracts were made. Brain lysates were fractionated on an SP HP column followed by NaCl gradient elution. Phosphorylating activities in fractions 7 and 8 (180–210 mM NaCl) were reduced by not eliminating. Phosphorylating activities in fractions 11 to 13 (300–360 mM NaCl) from Mst3 KO mouse brains contained strong AMPK and SIK3 phosphorylating activities. AMPK, AMP-activated protein kinase; HEK293T, human embryonic kidney 293T cell; LKB1, liver kinase B1; MST3, mammalian sterile 20–like kinase 3; SIK3, salt-inducible kinase 3.