Journal: The Journal of Biological Chemistry

Article Title: Identification of a reciprocal negative feedback loop between tau-modifying proteins MARK2 kinase and CBP acetyltransferase

doi: 10.1016/j.jbc.2022.101977

Figure Lengend Snippet: MARK2 modulates CBP acetyltransferase activity. A – C , soluble RIPA-extracted fractions were examined from 293A cells transfected with WT or catalytically inactive CBP (LD) variants in parallel with either MARK2-WT, constitutively active MARK2 (T208E, MARK2-TE), or catalytically inactive MARK2 (K82R, MARK2-KR) and immunoblotted with a pan-acetyl-lysine antibody ( A and B ). The relative band intensities of the soluble acetyl-lysine immunoreactivity in the presence of CBP-WT were quantified and plotted in ( C ). The graphs represent changes in acetyl-lysine immunoreactivity observed in the presence of MARK2-WT compared to control, as well as relative differences observed among the MARK2 variants. Error bars indicate SEM. n = 8 biologically independent experiments. p value was assessed by either unpaired Student’s t test ( left panel ) or one-way ANOVA with Tukey’s post hoc test ( right panel ). n.s. p > 0.05; ∗∗∗∗ p < 0.0001. D – F , RIPA-insoluble cell pellets were extracted with urea buffer to isolate the aggregated protein fraction from the same transfection described above in ( A ) and samples were similarly immunoblotted with a pan-acetyl-lysine antibody ( D and E ). The relative band intensities of insoluble acetyl-lysine immunoreactivity in the presence of CBP-WT were quantified and plotted in ( F ) similar to ( C ) above. Error bars indicate SEM. n = 8 biologically independent experiments. p value was assessed by either unpaired Student’s t test ( left panel ) or one-way ANOVA with Tukey’s post hoc test ( right panel ). ∗ p < 0.05; ∗∗∗∗ p < 0.0001. CBP, CREB-binding protein; MARK2, microtubule affinity-regulating kinase 2.

Article Snippet: The following primary antibodies were used in this study: acetyl-lysine (1:1000, Cell Signaling #9441), Myc (1:1000, Santa Cruz #sc-40), MARK2 (1:1000, Cell Signaling #9118), p-MARK2 (1:1000, Cell Signaling #4836), FLAG (1:1000, Sigma Aldrich #F1804), tau p-S262 (1:1000, Invitrogen #44-750G), total tau K9JA (1:5000, DAKO #A0024), total tau TAU-5 (1:1000, Invitrogen #AHB0042), CBP (1:1000, Cell Signaling #7389), and GAPDH (1:1000, Millipore #ABS16).

Techniques: Activity Assay, Transfection, Control, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Identification of a reciprocal negative feedback loop between tau-modifying proteins MARK2 kinase and CBP acetyltransferase

doi: 10.1016/j.jbc.2022.101977

Figure Lengend Snippet: CBP preferentially associates with inactive MARK2. A and B , coimmunoprecipitation (Co-IP) assays were performed to evaluate the binding of CBP to different MARK2 variants that show varying levels of kinase activity (MARK2-WT, MARK2-TE, MARK2-KR) in 293A cells by FLAG pull-down followed by immunoblotting to detect the total pool of myc-tagged MARK2 (Myc-MARK2). Input and IP samples are shown in ( A ) and ( B ) respectively. CBP preferentially interacts with the catalytically inactive MARK2 (MARK2-KR). C , Coomassie staining was used to detect immunoprecipitated FLAG-tagged CBP-WT in the absence (lane 1) or presence of different MARK2 variants (lanes 2–5) to illustrate a moderate reduction of CBP levels in the presence of active MARK2 (MARK2-WT and MARK2-TE). D and E , primary mouse cortical neurons at DIV14 were treated with or without 20 μM MARK2 inhibitor (39621) for 5 h. Neurons were then harvested and lysed, followed by immunoblotting with total CBP and GAPDH antibodies, and the increased CBP levels were quantified in (e). Error bars indicate SEM. n = 3 biologically independent experiments. p value was assessed by unpaired Student’s t test. ∗ p < 0.05. CBP, CREB-binding protein; MARK2, microtubule affinity-regulating kinase 2.

Article Snippet: The following primary antibodies were used in this study: acetyl-lysine (1:1000, Cell Signaling #9441), Myc (1:1000, Santa Cruz #sc-40), MARK2 (1:1000, Cell Signaling #9118), p-MARK2 (1:1000, Cell Signaling #4836), FLAG (1:1000, Sigma Aldrich #F1804), tau p-S262 (1:1000, Invitrogen #44-750G), total tau K9JA (1:5000, DAKO #A0024), total tau TAU-5 (1:1000, Invitrogen #AHB0042), CBP (1:1000, Cell Signaling #7389), and GAPDH (1:1000, Millipore #ABS16).

Techniques: Co-Immunoprecipitation Assay, Binding Assay, Activity Assay, Western Blot, Staining, Immunoprecipitation

Journal: The Journal of Biological Chemistry

Article Title: Identification of a reciprocal negative feedback loop between tau-modifying proteins MARK2 kinase and CBP acetyltransferase

doi: 10.1016/j.jbc.2022.101977

Figure Lengend Snippet: MARK2 is subjected to CBP-mediated lysine acetylation. A and B , recombinant WT MARK2 containing a GST tag (MARK2-WT) was incubated with recombinant WT CBP catalytic domain in the presence or absence of the cofactor acetyl-CoA, where indicated, in acetylation buffer for 1.5 h at 37 °C. The in vitro acetylation reaction was terminated by the addition of 6X SDS sample buffer and analyzed by immunoblotting using an acetyl-lysine antibody. Acetylated GST-tagged MARK2 is specifically detected at ∼ 120 kD, and the asterisk indicates acetylated CBP, which is known to be generated via CBP auto-acetylation. Acetyl-MARK2 levels were quantified and plotted in ( B ). Error bars indicate SEM. n = 3 biologically independent experiments. p value was assessed by one-way ANOVA with Tukey’s post hoc test. ∗∗∗∗ p < 0.0001. C and D , to confirm that MARK2 is acetylated in cells, a MARK2 immunoprecipitation was performed in 293A cells cotransfected with CBP and the various MARK2 variants. First, the presence of total CBP and MARK2 as inputs ( C ) were verified and the expected reduction in acetyl-lysine by active MARK2 was confirmed. Subsequently, the immunoprecipitated MARK2 fractions were blotted with the acetyl-lysine antibody to detect acetylated MARK2 ( D ) (see the prominent acetylated MARK2 protein band at ∼ 100 kDa). We note that the MARK2 expression plasmid used in the cell-based assays contains a myc tag and therefore migrates at ∼ 100 kD. CBP, CREB-binding protein; MARK2, microtubule affinity-regulating kinase 2.

Article Snippet: The following primary antibodies were used in this study: acetyl-lysine (1:1000, Cell Signaling #9441), Myc (1:1000, Santa Cruz #sc-40), MARK2 (1:1000, Cell Signaling #9118), p-MARK2 (1:1000, Cell Signaling #4836), FLAG (1:1000, Sigma Aldrich #F1804), tau p-S262 (1:1000, Invitrogen #44-750G), total tau K9JA (1:5000, DAKO #A0024), total tau TAU-5 (1:1000, Invitrogen #AHB0042), CBP (1:1000, Cell Signaling #7389), and GAPDH (1:1000, Millipore #ABS16).

Techniques: Recombinant, Incubation, In Vitro, Western Blot, Generated, Immunoprecipitation, Expressing, Plasmid Preparation, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Identification of a reciprocal negative feedback loop between tau-modifying proteins MARK2 kinase and CBP acetyltransferase

doi: 10.1016/j.jbc.2022.101977

Figure Lengend Snippet: PTM mapping identifies MARK2 as an acetylated substrate. A , 293A cells were transfected with a control vector (pcDNA5/TO) or MARK2 variants in the presence or absence of CBP-WT. Total cell lysates were extracted in low stringency NETN buffer, and myc-tagged MARK2 variants were immunopurified using myc antibodies conjugated to protein A/G beads. The Coomassie-stained gel shows the purified MARK2 protein at ∼ 100 kD that was subsequently gel-excised and analyzed by LC-MS/MS. B , a MARK2 molecular domain schematic illustrates the acetylated lysines (K) in green , phosphorylated serines (S) in blue and phosphorylated threonines (T) in red . In the presence of CBP-WT, a panel of MARK2 acetylation sites were identified and the peptide properties are described in Table S1 . C , in vitro kinase reactions were performed in the presence or absence of MARK2-WT, CBP-WT, full-length tau-WT (2N4R), and acetyl-CoA, followed by immunoblotting analysis to determine whether MARK2 acetylation altered tau phosphorylation at residue S262. D , quantification of p-S262 across all conditions was determined by band densitometry. Error bars indicate SEM. n = 3 biologically independent experiments. p value was assessed by one-way ANOVA with Tukey’s post hoc test. n.s. p > 0.05; ∗ p < 0.05. CBP, CREB-binding protein; MARK2, microtubule affinity-regulating kinase 2; PTM, posttranslational modification.

Article Snippet: The following primary antibodies were used in this study: acetyl-lysine (1:1000, Cell Signaling #9441), Myc (1:1000, Santa Cruz #sc-40), MARK2 (1:1000, Cell Signaling #9118), p-MARK2 (1:1000, Cell Signaling #4836), FLAG (1:1000, Sigma Aldrich #F1804), tau p-S262 (1:1000, Invitrogen #44-750G), total tau K9JA (1:5000, DAKO #A0024), total tau TAU-5 (1:1000, Invitrogen #AHB0042), CBP (1:1000, Cell Signaling #7389), and GAPDH (1:1000, Millipore #ABS16).

Techniques: Transfection, Control, Plasmid Preparation, Staining, Purification, Liquid Chromatography with Mass Spectroscopy, In Vitro, Western Blot, Phospho-proteomics, Residue, Binding Assay, Modification

Journal: The Journal of Biological Chemistry

Article Title: Identification of a reciprocal negative feedback loop between tau-modifying proteins MARK2 kinase and CBP acetyltransferase

doi: 10.1016/j.jbc.2022.101977

Figure Lengend Snippet: CBP-mediated acetylation regulates MARK2 kinase activity. A – C , primary mouse cortical neurons were dissected, plated, and transduced at DIV3 with a lentivirus expressing either control (empty lentivirus), CBP-NES-WT (cytoplasmic CBP), or CBP-NES-LD (cytoplasmic inactive CBP). Neurons were harvested at DIV10 and analyzed by immunoblotting with MARK2, p-S262 tau, total tau, FLAG, and acetyl-lysine antibodies, while GAPDH served as a loading control. The extent of p-S262 and MARK2 were quantified and plotted in ( B ) and ( C ), which showed that tau phosphorylation at S262 is reduced in the presence of CBP-NES-WT. Error bars indicate SEM; n = 3 biologically independent experiments. p value was assessed by one-way ANOVA with Tukey’s post hoc test. ∗ p < 0.05. D , gel-excised MARK2-WT was immunopurified from cell lysates, and global PTM site mapping was performed using LC-MS/MS. Purified MARK2 was reduced with DTT, alkylated with iodoacetamide, and digested in-gel with trypsin overnight, then subjected to LC-MS/MS analysis. MS/MS spectrum of the triply-charged ion at m/z 726.6586 corresponding to MARK2 peptide LD t F c GSPPYAAPELFQGK is shown. The spectrum provides evidence that residue T208 is in fact phosphorylated (an indicator of MARK2 activation status). E and F , 293A cells transfected with WT or catalytically inactive (LD) CBP variants in parallel with the kinase inactive variant of MARK2 (MARK2-KR) were immunoblotted with p-MARK2 (T208) or total MARK2 antibodies to analyze T208 phosphorylation status in response to acetylation. The ratio of P-MARK2 to total MARK2 (relative p-MARK2) was quantified and plotted in (f). Error bars indicate SEM; n = 6 biologically independent experiments. p value was assessed by unpaired Student’s t test. ∗ p < 0.05. CBP, CREB-binding protein; MARK2, microtubule affinity-regulating kinase 2; NES, nuclear export signal; PTM, posttranslational modification.

Article Snippet: The following primary antibodies were used in this study: acetyl-lysine (1:1000, Cell Signaling #9441), Myc (1:1000, Santa Cruz #sc-40), MARK2 (1:1000, Cell Signaling #9118), p-MARK2 (1:1000, Cell Signaling #4836), FLAG (1:1000, Sigma Aldrich #F1804), tau p-S262 (1:1000, Invitrogen #44-750G), total tau K9JA (1:5000, DAKO #A0024), total tau TAU-5 (1:1000, Invitrogen #AHB0042), CBP (1:1000, Cell Signaling #7389), and GAPDH (1:1000, Millipore #ABS16).

Techniques: Activity Assay, Expressing, Control, Western Blot, Phospho-proteomics, Liquid Chromatography with Mass Spectroscopy, Purification, Tandem Mass Spectroscopy, Residue, Activation Assay, Transfection, Variant Assay, Binding Assay, Modification

Journal: The Journal of Biological Chemistry

Article Title: Identification of a reciprocal negative feedback loop between tau-modifying proteins MARK2 kinase and CBP acetyltransferase

doi: 10.1016/j.jbc.2022.101977

Figure Lengend Snippet: MARK2 protein levels are reduced in tauopathy mice and human AD. A and B , the cortex was isolated from 12-month-old WT and PS19 mice and extracted using high-salt buffer. High-salt fractions were analyzed by immunoblotting using MARK2 or total tau (TAU-5) antibodies. The relative levels of MARK2 protein were quantified in ( B ). Error bars indicate SEM. n = 3 biologically independent experiments. p value was assessed by unpaired Student’s t test. ∗ p < 0.05. C and D , high-salt soluble and urea-extracted insoluble fractions of cortical brain homogenates from control or AD brain tissue was analyzed by immunoblotting using MARK2 or total tau (TAU-5) antibodies. The urea-extracted tau is present only in AD brain, confirming the presence of tau pathology. GAPDH served as a loading control. The levels of MARK2 in the high-salt fraction were quantified in ( D ). The molecular weight of MARK2 is ∼ 77 to 90 kD, corresponding to the protein doublets observed in mouse and human tissues. For full data transparency, all protein bands are displayed including possible MARK2 breakdown products < 75 kDa. Error bars indicate SEM. n = 3 biologically independent experiments. p value was assessed by unpaired Student’s t test. ∗ p < 0.05. AD, Alzheimer’s disease; MARK2, microtubule affinity-regulating kinase 2.

Article Snippet: The following primary antibodies were used in this study: acetyl-lysine (1:1000, Cell Signaling #9441), Myc (1:1000, Santa Cruz #sc-40), MARK2 (1:1000, Cell Signaling #9118), p-MARK2 (1:1000, Cell Signaling #4836), FLAG (1:1000, Sigma Aldrich #F1804), tau p-S262 (1:1000, Invitrogen #44-750G), total tau K9JA (1:5000, DAKO #A0024), total tau TAU-5 (1:1000, Invitrogen #AHB0042), CBP (1:1000, Cell Signaling #7389), and GAPDH (1:1000, Millipore #ABS16).

Techniques: Isolation, Western Blot, Control, Molecular Weight