Activation of a GST-tagged AKT2/PKBβ

Baer, Kristin; Lisinski, Ivonne; Gompert, Monika; Stuhlmann, Dominik; Schmolz, K.; Klein, Helmut W.; Al-Hasani, Hadi

doi:10.1016/j.bbagen.2005.04.002

Cited by 6 publications

(11 citation statements)

References 34 publications

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“…We were able to obtain approximately 1 mg of purified PKBα protein from 20 larvae. Literatures reported that the yields of GST-PKBα (kinase domain) and GST-PKBβ (kinase domain) were 32 mg and 5–15 mg/Liter culture, respectively3233. Of note, these values are reported as GST-fusion proteins.…”

Section: Discussionmentioning

confidence: 99%

Efficient and cost effective production of active-form human PKB using silkworm larvae

Maesaki

Satoh

Taoka

et al. 2014

Sci Rep

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Protein kinase B (PKB) also known as Akt is involved in many signal transduction pathways. As alterations of the PKB pathway are found in a number of human malignancies, PKB is considered an important drug target for cancer therapy. However, production of sufficient amounts of active PKB for biochemical and structural studies is very costly because of the necessity of using a higher organism expression system to obtain phosphorylated PKB. Here, we report efficient production of active PKBα using the BmNPV bacmid expression system with silkworm larvae. Following direct injection of bacmid DNA, recombinant PKBα protein was highly expressed in the fat bodies of larvae, and could be purified using a GST-tag and then cleaved. A final yield of approximately 1 mg PKBα/20 larvae was recorded. Kinase assays showed that the recombinant PKBα possessed high phosphorylation activity. We further confirmed phosphorylation on the activation loop by mass spectrometric analysis. Our results indicate that the silkworm expression system is of value for preparation of active-form PKBα with phosphorylation on the activation loop. This efficient production of the active protein will facilitate further biochemical and structural studies and stimulate subsequent drug development.

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Section: Discussionmentioning

confidence: 99%

Efficient and cost effective production of active-form human PKB using silkworm larvae

Maesaki

Satoh

Taoka

et al. 2014

Sci Rep

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“…The baculovirus expression vector pAcGS2 was purchased from BD Bioscience (Heidelberg, Germany). A His 6 -tagged cDNA fragment for the long mouse isoform of Tbc1d1 (1255 amino acids; NP_001297540.1) was cloned into pAcGS2 to generate a baculovirus as described (37). A baculovirus for a GST fusion protein of mouse Rab10 was constructed as described (37).…”

Section: Dna Constructsmentioning

confidence: 99%

“…A His 6 -tagged cDNA fragment for the long mouse isoform of Tbc1d1 (1255 amino acids; NP_001297540.1) was cloned into pAcGS2 to generate a baculovirus as described (37). A baculovirus for a GST fusion protein of mouse Rab10 was constructed as described (37). The constructs for TBC1D1-R854K and -R125W mutations were generated using PCR-based QuikChange Site-directed Mutagenesis Kit (Agilent Technologies, CA).…”

Section: Dna Constructsmentioning

confidence: 99%

“…GST-Rab8a, GST-Rab14, GST-14-3-3, GST-TBC1D1 GAP domain, and GST-PTBD domains were expressed in E. coli BL21-CodonPlus (DE3)-RIL and purified by GSH affinity chromatography (GE Healthcare) as described (26,39). GST-Rab10 was expressed via the baculovirus system and purified as described (37).…”

Section: Protein Expression and Purificationmentioning

confidence: 99%

“…Recombinant human constitutively active AKT2 was purified from Sf9 lysates as described previously (37) and purified trimeric AMPK was obtained from Invitrogen. Two l of the purified enzyme (about 4 pmol of protein) were used to phosphorylate about 2 pmol of purified TBC1D1 for 20 min at room temperature in the presence of 2 mM ATP, 40 mM Tris-HCl, pH Regulation of recombinant TBC1D1 in vitro 7.4, 8 mM MgCl 2 , and 0.4 mM dithiothreitol (DTT) in a volume of 100 l. Two microunits of recombinant human AMPK was used for the phosphorylation of TBC1D1 under similar conditions, with the exception of adding 200 M AMP.…”

Section: Kinase Assaysmentioning

confidence: 99%

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AKT and AMP-activated protein kinase regulate TBC1D1 through phosphorylation and its interaction with the cytosolic tail of insulin-regulated aminopeptidase IRAP

Mafakheri

Flörke

Kanngießer

et al. 2018

Journal of Biological Chemistry

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In skeletal muscle, the Rab GTPase-activating (GAP) protein TBC1D1 is phosphorylated by AKT and AMP-activated protein kinase (AMPK) in response to insulin and muscle contraction. Genetic ablation of Tbc1d1 or mutation of distinct phosphorylation sites impairs intracellular GLUT4 retention and GLUT4 traffic, presumably through alterations of the activation state of downstream Rab GTPases. Previous studies have focused on characterizing the C-terminal GAP domain of TBC1D1 that lacks the known phosphorylation sites, as well as putative regulatory domains. As a result, it has been unclear how phosphorylation of TBC1D1 would regulate its activity. In the present study, we have expressed, purified, and characterized recombinant full-length TBC1D1 in Sf9 insect cells via the baculovirus system. Full-length TBC1D1 showed RabGAP activity toward GLUT4-associated Rab8a, Rab10, and Rab14, indicating similar substrate specificity as the truncated GAP domain. However, the catalytic activity of the full-length TBC1D1 was markedly higher than that of the GAP domain. Although in vitro phosphorylation of TBC1D1 by AKT or AMPK increased 14-3-3 binding, it did not alter the intrinsic RabGAP activity. However, we found that TBC1D1 interacts through its N-terminal PTB domains with the cytoplasmic domain of the insulin-regulated aminopeptidase, a resident protein of GLUT4 storage vesicles, and this binding is disrupted by phosphorylation of TBC1D1 by AKT or AMPK. In summary, our findings suggest that other regions outside the GAP domain may contribute to the catalytic activity of TBC1D1. Moreover, our data indicate that recruitment of TBC1D1 to GLUT4-containing vesicles and not its GAP activity is regulated by insulin and contraction-mediated phosphorylation.

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Sphingomyelin-rich domains are sites of lysenin oligomerization: Implications for raft studies

Kulma

Hereć

Grudziński

et al. 2010

Biochimica et Biophysica Acta (BBA) - Biomembranes

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Lysenin is a self-assembling, pore-forming toxin which specifically recognizes sphingomyelin. Mutation of tryptophan 20 abolishes lysenin oligomerization and cytolytic activity. We studied the interaction of lysenin WT and W20A with sphingomyelin in membranes of various lipid compositions which, according to atomic force microscopy studies, generated either homo- or heterogeneous sphingomyelin distribution. Liposomes composed of SM/DOPC, SM/DOPC/cholesterol and SM/DPPC/cholesterol could bind the highest amounts of GST-lysenin WT, as shown by surface plasmon resonance analysis. These lipid compositions enhanced the release of carboxyfluorescein from liposomes induced by lysenin WT, pointing to the importance of heterogeneous sphingomyelin distribution for lysenin WT binding and oligomerization. Lysenin W20A bound more weakly to sphingomyelin-containing liposomes than did lysenin WT. The same amounts of lysenin W20A bound to sphingomyelin mixed with either DOPC or DPPC, indicating that the binding was not affected by sphingomyelin distribution in the membranes. The mutant lysenin had a limited ability to penetrate hydrophobic region of the membrane as indicated by measurements of surface pressure changes. When applied to detect sphingomyelin on the cell surface, lysenin W20A formed large conglomerates on the membrane, different from small and regular clusters of lysenin WT. Only lysenin WT recognized sphingomyelin pool affected by formation of raft-based signaling platforms. During fractionation of Triton X-100 cell lysates, SDS-resistant oligomers of lysenin WT associated with membrane fragments insoluble in Triton X-100 while monomers of lysenin W20A partitioned to Triton X-100-soluble membrane fractions. Altogether, the data suggest that oligomerization of lysenin WT is a prerequisite for its docking in raft-related domains.

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Activation of a GST-tagged AKT2/PKBβ

Cited by 6 publications

References 34 publications

Efficient and cost effective production of active-form human PKB using silkworm larvae

Efficient and cost effective production of active-form human PKB using silkworm larvae

AKT and AMP-activated protein kinase regulate TBC1D1 through phosphorylation and its interaction with the cytosolic tail of insulin-regulated aminopeptidase IRAP

Sphingomyelin-rich domains are sites of lysenin oligomerization: Implications for raft studies

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