Phosphorylation of LKB1 by PDK1 Inhibits Cell Proliferation and Organ Growth by Decreased Activation of AMPK

Borkowsky, Sarah; Gass, Maximilian; Alavizargar, Azadeh; Hanewinkel, Johannes; Hallstein, Ina; Nedvetsky, Pavel I.; Heuer, Andreas; Krahn, Michael P.

doi:10.3390/cells12050812

Cited by 5 publications

(6 citation statements)

References 72 publications

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“…In summary, we revealed that the polybasic motif of LKB1 can establish stable, albeit highly dynamic, binding with membranes with low amounts of PA. This suggests that farnesylation of LKB1 is not specifically required for stable association of LKB1 with membranes, which has been supported by in vivo experiments (Dogliotti et al 13,35 ). Furthermore, the LKB1-membrane interaction revealed interesting mutual effects of the protein and anionic lipids, such as proteins' structural modifications and agglomeration of PAs, providing microscopic and thermodynamic insights into the interaction of LKB1 with membranes containing PAs.…”

Section: Discussionmentioning

confidence: 67%

“…Schneider S2R+ cells on coverslips were transfected with GFP-LKB1 variants under a ubiquitous promoter (Ubi::GFP-LKB1) using FUGENE (Promega). 35 48 h after transfection, cells were fixed with 4% paraformaldehyde in PBS pH7.4 for 20min. Subsequently, cells were washed three times with PBS and nuclei were labelled with DAPI for 20min.…”

Section: Cell Culture and Transfectionmentioning

confidence: 99%

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Elucidating the Membrane Binding Process of a Disordered Protein: Dynamic Interplay of Anionic Lipids and the Polybasic Region

Alavizargar

Gass

Krahn

et al. 2023

Preprint

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Liver kinase LKB1 is a serine/threonine kinase, which functions as a master kinase, activating several downstream kinases. Thereby, it is implicated in many cellular processes such as cell proliferation and cell polarity. Moreover, LKB1 regulates cell cycle progression and apoptosis and is mutated or downregulated in various types of cancer. LKB1 itself becomes fully activated upon recruitment to the plasma membrane by binding of its C-terminal polybasic motif consisting of eight Lysines/Arginines to phospholipids. Here we present extensive molecular dynamics (MD) simulations of the C-terminus of LKB1 containing the polybasic motif interacting with a model membrane composed of phosphatidylcholin (POPC) and phosphatidic acid (PA). Protein-membrane binding effects are due to the electrostatic interactions between the polybasic amino acids and PA lipids. For significant binding the first three lysines turn out to be dispensable, which was also recapitulated in cell culture using transfected GFP-LKB1 variants. LKB1-membrane binding results in a structural change of the protein as well as of the membrane, in particular accumulation of PA lipids and reduced thickness at the protein-membrane contact area. The protein-lipid binding turns out to be highly dynamic due to an interplay of PA-PA repulsion and protein-PA attraction. The thermodynamics of this interplay is captured by a statistical fluctuation model, which allows the estimation of both energies. Quantification of the significance of each polar amino acid in the polybasic provides detailed insights into the molecular mechanism of the protein-membrane binding of LKB1. The results likely can be transferred to other proteins, which interact by intrinsically disordered polybasic regions with anionic membranes.

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Section: Discussionmentioning

confidence: 67%

Section: Cell Culture and Transfectionmentioning

confidence: 99%

Elucidating the Membrane Binding Process of a Disordered Protein: Dynamic Interplay of Anionic Lipids and the Polybasic Region

Alavizargar

Gass

Krahn

et al. 2023

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

“…In summary, we revealed that the polybasic motif of LKB1 can establish stable, albeit highly dynamic, binding with membranes with low amounts of PA. This suggests that farnesylation of LKB1 is not specifically required for stable association of LKB1 with membranes, which has been supported by in vivo experiments (Dogliotti et al 13,40 ). Furthermore, the LKB1-membrane interaction revealed interesting mutual effects of the protein and anionic lipids, such as proteins' structural modifications and agglomeration of PAs, providing microscopic and thermodynamic insights into the interaction of LKB1 with membranes containing PAs.…”

Section: ■ Conclusionmentioning

confidence: 67%

“…Schneider S2R+ cells on coverslips were transfected with GFP-LKB1 variants under a ubiquitous promoter (Ubi::GFP-LKB1) using FUGENE (Promega). 40 48 h after transfection, cells were fixed with 4% paraformaldehyde in PBS pH 7.4 for 20 min. Subsequently, cells were washed three times with PBS, and nuclei were labeled with DAPI for 20 min.…”

Section: Cell Culture and Transfectionmentioning

confidence: 99%

Elucidating the Membrane Binding Process of a Disordered Protein: Dynamic Interplay of Anionic Lipids and the Polybasic Region

Alavizargar,

Gass,

Krahn

et al. 2024

ACS Phys. Chem Au

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Intrinsically disordered regions of proteins are responsible for many biological processes such as in the case of liver kinase B1 (LKB1)�a serine/threonine kinase relevant for cell proliferation and cell polarity. LKB1 becomes fully activated upon recruitment to the plasma membrane by binding of its disordered C-terminal polybasic motif consisting of eight lysines/arginines to phospholipids. Here, we present extensive molecular dynamics (MD) simulations of the polybasic motif interacting with a model membrane composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleyl phosphatidic acid (PA) and cell culture experiments. Protein−membrane binding effects are due to the electrostatic interactions between the polybasic amino acids and PAs. For significant binding, the first three lysines turn out to be dispensable, which was also recapitulated in cell culture using transfected GFP-LKB1 variants. LKB1−membrane binding results in nonmonotonous changes in the structure of the protein as well as the membrane, in particular, accumulation of PAs and reduced thickness at the protein−membrane contact area. The protein−lipid binding turns out to be highly dynamic due to an interplay of PA−PA repulsion and protein−PA attraction. The thermodynamics of this interplay is captured by a statistical fluctuation model, which allows the estimation of both energies. Quantification of the significance of each polar amino acid in the polybasic provides detailed insights into the molecular mechanism of protein−membrane binding of LKB1. These results can likely be transferred to other proteins, which interact by intrinsically disordered polybasic regions with anionic membranes.

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“…The prediction result of phosphorylation sites showed that the PTHLH protein may contain phosphorylation sites for protein kinases such as PKA, PKC, PKG and p38 MAPK. Protein phosphorylation, as a widespread reversible post-translational modification in biology, is involved in gene expression regulation, signal transduction, and is closely related to cell proliferation, apoptosis and development of cancer [22][23][24][25]. Inhibitors of p38 MAPK, PKC can reduce synthesis and secretion of PTHLH, speculating that PTHLH protein may be affected by the comprehensive action of various kinases to influence protein phosphorylation and its activity, thereby regulating the expression of the…”

Section: Discussionmentioning

confidence: 99%

The spatio-temporal expression analysis of parathyroid hormone like hormone gene provides a new insight for bone growth of the antler tip tissue in sika deer

Xing,

Han,

Wang

et al. 2024

Anim Biosci

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Title of the manuscript: The spatio-temporal expression analysis of parathyroid hormone like hormone gene provide a new insight for bone growth of the antler tip tissue in sika deer ABSTRACT Objective: Parathyroid hormone like hormone (PTHLH), as an essential factor for bone growth, is involved in a variety of physiological processes. The aim of this study was to explore the role of PTHLH gene in the growth of antler. Methods: The coding sequence (CDS) of PTHLH gene cDNA was obtained by cloning in sika deer (Cervus nippon), and the bioinformatics was analyzed. The quantitative real-time polymerase chain reaction (qRT-PCR) was used to analyze the differences expression of PTHLH mRNA in different tissues of the antler tip at different growth periods (early period, EP; middle period, MP; late period, LP). Results:The CDS of PTHLH gene was 534bp in length and encoded 177 amino acids.Predictive analysis results revealed that the PTHLH protein was a hydrophilic protein without transmembrane structure, with its secondary structure consisting mainly of random coil. The PTHLH protein of sika deer had the identity of 98.31%, 96.82%, 96.05% and 94.92% with Cervus canadensis, Bos mutus, Oryx dammah and Budorcas taxicolor, which were highly conserved among the artiodactyls. The qRT-PCR results showed that PTHLH mRNA had a unique spatio-temporal expression pattern in antler.In the dermis, precartilage, and cartilage tissues, the expression of PTHLH mRNA was extremely significantly higher in MP than in EP, LP (p<0.01). In the mesenchyme A c c e p t e d A r t i c l e 5 tissue, the expression of PTHLH mRNA in MP was significantly higher than that of EP (p<0.05), but extremely significantly lower than that of LP (p<0.01). The expression of PTHLH mRNA in antler tip tissues at all growth periods had approximately the same trend, that is, from distal to basal, it was first down-regulated from the dermis to the mesenchyme and then continuously up-regulated to the cartilage tissue. Conclusion:PTHLH gene may promote the rapid growth of antler mainly through its extensive regulatory effect on the antler tip tissue.

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Phosphorylation of LKB1 by PDK1 Inhibits Cell Proliferation and Organ Growth by Decreased Activation of AMPK

Cited by 5 publications

References 72 publications

Elucidating the Membrane Binding Process of a Disordered Protein: Dynamic Interplay of Anionic Lipids and the Polybasic Region

Elucidating the Membrane Binding Process of a Disordered Protein: Dynamic Interplay of Anionic Lipids and the Polybasic Region

Elucidating the Membrane Binding Process of a Disordered Protein: Dynamic Interplay of Anionic Lipids and the Polybasic Region

The spatio-temporal expression analysis of parathyroid hormone like hormone gene provides a new insight for bone growth of the antler tip tissue in sika deer

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