Fig 1: CaMKII phosphorylated Rab4a and regulated its functions in NIH-3T3 cells Immunoprecipitation (IP) and western blot (WB) experiments in cells expressing GFP-Rab4a fusion protein showing that AAS did not change the interaction between Rab4a and GDI. H/chain, Ig heavy chain. Normal IgG was used as negative control for IP.Double fluorescence labeling experiment showing that AAS had no effect on Rab4a-GDI co-localization (experiment repeated 3 times).Sequence alignment of the putative CaMKII substrate site (Thr137) in Rab4a across different species. In vitro phosphorylation experiments using FLAG-tagged wild type (WT) and T137A mutant of Rab4a(120-154) peptide showing that CaMKII could phosphorylate the wild-type peptide (experiment repeated 2 times). p-(S/T), anti-phospho serine/threonine antibody.Representative results of IP/WB experiments in NIH-3T3 cells expressing wild-type GFP-Rab4a or GFP-Rab4a-T137A, showing that AAS increased the phosphorylation of wild-type Rab4a but not Rab4a-T137A.Representative results of IP/WB experiments showing that AAS increased the phosphorylation level of endogenous Rab4a, and this response was abolished by KN-93 pretreatment.Representative fluorescence microscopy images showing that in the cells with a high level of Rab4a-T137A expression (arrows), AAS failed to stimulate DiI-LDL endocytosis, while in cells with poor Rab4a-T137A expression (asterisk), AAS-stimulated DiI-LDL endocytosis was retained.Surface protein biotinylation assay showing that Rab4a-T137A overexpression diminished the effect of AAS on LDLR accumulation on PM (example from 3 independent experiments with similar results).Western blots and quantitative data showing that treatment with the serine/threonine protein phosphatase inhibitor okadaic acid (OA, 0.5 µM for 60 min) increased the phosphorylation level of CaMKII (Thr286) in nonstressed cells.Fluorescence microscopy data showing that okadaic acid treatment in nonstressed cells mimicked the effect of AAS on DiI-LDL endocytosis.Double fluorescence labeling and fluorescence intensity profile analysis showing that okadaic acid treatment in nonstressed cells increased LDLR-Rab4a co-localization in the cell periphery (arrowheads) (experiment repeated 3 times). Dotted lines indicated the sampling location for fluorescence intensity profile analysis.Representative IP/WB results showing that in cells overexpressing GFP-Rab4a fusion proteins, AAS increased Rabenosyn-5 binding with wild-type Rab4a, but not that with Rab4a-T137A. The stimulating effect of AAS on Rabenosyn-5/Rab4a binding was blunted in the presence of KN-93. Data information: Data were mean ± SD. *P < 0.05, one-way ANOVA or unpaired t-test as appropriate. Source data are available online for this figure.
Fig 2: AAS-stimulated LDL endocytosis was dependent on Ca2+ and CaMKII signaling Fluorescence microscopy data showing that knocking down GCN2 expression with siRNA did not modify AAS-stimulated DiI-LDL endocytosis.Fluorescence microscopy data showing that pretreatment of the cells with the GCN2 inhibitor GCN2iB had no effect on AAS-stimulated DiI-LDL endocytosis.Measurement of intracellular Ca2+ concentration using fluorescent confocal microscopy in living cells incubated in AAS medium and medium with amino acids replenished, respectively (mean data of n = 3 independent experiments).Fluorescence microscopy data showing that pretreating cells with the Ca2+ chelator BAPTA-AM (5 µM) abolished the effect of AAS on DiI-LDL endocytosis. DMSO was vehicle control.Flow cytometry analysis showing that BAPTA-AM pretreatment abolished AAS-induced increase in the cell surface LDLRs (example from 3 independent experiments with similar results).Fluorescence microscopy data showing that treatment with the calcium ionophore A23187 (2 µM for 120 min) enhanced DiI-LDL endocytosis in the cells maintained in complete medium.Flow cytometry analysis showing that A23187 produced a comparable effect as AAS on the abundance of the cell surface LDLRs in the cells maintained in complete medium (experiment repeated 3 times).Double fluorescence labeling and fluorescence intensity profile analysis showing that A23187 increased LDLR-Rab4a co-localization in the cell periphery (arrowheads) (example from 3 independent experiments with similar results). Dotted lines indicated the sampling location for fluorescence intensity profile analysis. DMSO was vehicle control.Western blots and densitometry data showing that AAS increased the level of CaMKII phosphorylation (Thr286).Fluorescence microscopy images and quantitative data showing that pretreatment with the CaMKII inhibitor KN-93 (5 µM), but not the calcium/calmodulin-dependent kinase kinase inhibitor STO-609 (5 µM), abolished AAS-stimulated DiI-LDL endocytosis.Flow cytometry data showing that KN-93, but not STO-609, abolished AAS-stimulated increase in the cell surface LDLR abundance (experiment repeated 3 times). Experiments in A were performed in HepG2 cells; other experiments were in NIH-3T3 cells. Data information: Data were mean ± SD. *P < 0.05, one-way ANOVA or unpaired t-test as appropriate. NS, no significance. Source data are available online for this figure.
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