Journal: Cancers

Article Title: SHED-Dependent Oncogenic Signaling of the PEAK3 Pseudo-Kinase

doi: 10.3390/cancers13246344

Figure Lengend Snippet: PEAK3 interactomics identified GRB2, ASAP1/2, and PYK2 as SHED-dependent binders. ( a ) Interactome of PEAK3 in U2OS cells represented as volcano plots of the MS data ( n = 3). ( b ) and ( c ) Validation by western blot analysis of PEAK3 interaction with the indicated signaling proteins in U2OS ( b ) and THP1 cells ( c ) ( n = 2). The relative levels of PYK2, ASAP1, and GRB2 associated with ST-PEAK3 WT and AE mutant are shown.

Article Snippet: The pCDNA3.1-V5-PTK2B (PYK2) plasmid was a gift from Kai Johnsson (Addgene plasmid # 127233).

Techniques: Biomarker Discovery, Western Blot, Mutagenesis

Journal: Cancers

Article Title: SHED-Dependent Oncogenic Signaling of the PEAK3 Pseudo-Kinase

doi: 10.3390/cancers13246344

Figure Lengend Snippet: PEAK3 activates PYK2 signaling. ( a ) PYK2 activation by PEAK3 in HEK293T cells. The levels of protein tyrosine phosphorylation, PEAK3, PYK2, and phosphorylated PYK2 (pPYK2, pTyr402, and pTyr881) were assessed in cells transfected with the indicated constructs for 40 h (left). Relative quantification of PYK2 activation by PEAK3 (mean ± SD; n = 3); ** p < 0.01 (Student’s t test) (right). ( b ) PEAK3 tyrosine phosphorylation and its association with GRB2 and ASAP1 by PYK2 in HEK293T cells. The level-indicated proteins and their tyrosine phosphorylation in the PEAK3 pull-down were assessed in cells transfected with indicated constructs for 40 h. Relative quantification of PEAK3 tyrosine phosphorylation and its association with GRB2 and ASAP1 (mean ± SD; n = 3); ** p < 0.01, *** p < 0.001 (Student’s t test) (right). ( c ) PEAK3 tyrosine phosphorylation by PYK2 in THP1 cells. The pull-down of PEAK3 and its associated proteins were assessed in THP1 cells overexpressing PEAK3 and treated or not with pervanadate (PV) (0.1 μM for 15 min) or PYK2i (1 μM for 2h) as indicated. ( d ) Relative quantification of PEAK3 tyrosine phosphorylation and its association with GRB2 and ASAP1 in THP1 cells (mean ± SD; n = 3); * p < 0.05, ** p < 0.01 (Student’s t test) (right). ( e ) Increased ASAP1 tyrosine phosphorylation by PEAK3 expression in a PYK2-dependent manner. Immunoprecipitated ASAP1 tyrosine phosphorylation and its association with ST-PEAK3 from indicated THP1 cells treated as described in c ( n = 2).

Article Snippet: The pCDNA3.1-V5-PTK2B (PYK2) plasmid was a gift from Kai Johnsson (Addgene plasmid # 127233).

Techniques: Activation Assay, Phospho-proteomics, Transfection, Construct, Quantitative Proteomics, Expressing, Immunoprecipitation

Journal: Cancers

Article Title: SHED-Dependent Oncogenic Signaling of the PEAK3 Pseudo-Kinase

doi: 10.3390/cancers13246344

Figure Lengend Snippet: PYK2 activity mediates PEAK3–AKT signaling. ( a ) PYK2 activation by PEAK3 overexpressing U2OS cells that were serum-starved or not for >20 h. Western blots of the levels of PEAK3, PYK2, and pPYK2 ( n = 2). ( b ) Regulation of PYK2 activity by endogenous PEAK3 in THP1 cells. PYK2 activity (pTyr402 PYK2 level) was measured in cells transfected with the indicated siRNAs. Quantification of PYK2 activity (mean ± SD; n = 3); * p < 0.05 (Student’s t test) (bottom). ( c ) PYK2 inhibition reduces PEAK3 activation of AKT in the absence of growth factors. U2OS cells that express or do not express PEAK3 were serum starved overnight or not, in the presence of PYK2 inhibitor (PYK2i) (1 μM) or vehicle (DMSO) as indicated. Western blots of the level of PEAK3, AKT, and pAKT (left) and relative quantification of AKT activity (mean ± SD; n = 3); ** p < 0.01 (Student’s t test) (right).

Article Snippet: The pCDNA3.1-V5-PTK2B (PYK2) plasmid was a gift from Kai Johnsson (Addgene plasmid # 127233).

Techniques: Activity Assay, Activation Assay, Western Blot, Transfection, Inhibition, Quantitative Proteomics

Journal: Journal of Alzheimer's Disease

Article Title: Pyk2 is a Novel Tau Tyrosine Kinase that is Regulated by the Tyrosine Kinase Fyn

doi: 10.3233/JAD-180054

Figure Lengend Snippet: Antibody applications

Article Snippet: Pyk2-V5 and Pyk2-GFP vectors were generated by cloning the Pyk2 cDNA into pcDNA6.2/V5-DEST and pcDNA6.2/N-EmGFP-DEST vectors (Gateway cloning, Thermo Fisher Invitrogen).

Techniques:

Journal: Journal of Alzheimer's Disease

Article Title: Pyk2 is a Novel Tau Tyrosine Kinase that is Regulated by the Tyrosine Kinase Fyn

doi: 10.3233/JAD-180054

Figure Lengend Snippet: Pyk2 enhances tau pathology in vivo . A) The murine Thy1.2 promoter drives human P301L tau and FLAG-tagged Pyk2 expression in pR5 and Pyk2 transgenic mice, respectively. Representative immunofluorescence staining of brain sections from 12–15-month-old pR5 and double transgenic pR5/Pyk2 animals shown in (B) with magnified view in (C). Scale bars represent 500 μ m (B) and 50 μ m (C). D) Quantification of the pY18-tau positive area in the hippocampus of transgenic brains. Mean±s.e.m, n = 7 mice per group, two-tailed t test, ** p < 0.01. E) Validation of pY18-tau antibody using immunoblots of a lysate from human tau-expressing HEK cells. WT, wild-type; Y18A, tyrosine to alanine mutant. F) Representative immunoblots of sequentially (RAB/RIPA) fractionated hippocampal homogenates from 12–15-month-old pR5 and pR5/Pyk2 mice. Both pY18-tau and HT7 antibodies react with transgenic human tau but not endogenous mouse tau. ‘h’ indicates human transgenic tau, and ‘m’ indicates endogenous murine tau. G) Quantification of immunoblots in (F). Fold changes are relative to the pR5 group. Mean±s.e.m, n = 4–6 mice per group, two-tailed t test, # p = 0.075, * p < 0.05.

Article Snippet: Pyk2-V5 and Pyk2-GFP vectors were generated by cloning the Pyk2 cDNA into pcDNA6.2/V5-DEST and pcDNA6.2/N-EmGFP-DEST vectors (Gateway cloning, Thermo Fisher Invitrogen).

Techniques: In Vivo, Expressing, Transgenic Assay, Immunofluorescence, Staining, Two Tailed Test, Western Blot, Mutagenesis

Journal: Journal of Alzheimer's Disease

Article Title: Pyk2 is a Novel Tau Tyrosine Kinase that is Regulated by the Tyrosine Kinase Fyn

doi: 10.3233/JAD-180054

Figure Lengend Snippet: Localization and interaction of Pyk2 and tau. A) Representative immunofluorescence staining of Pyk2 in primary hippocampal neurons (DIV (days in vitro ) 10) cultured in microfluidic chambers. Cells were counterstained for tau (all compartments including axons) and MAP2 (all compartments excluding axons). DAPI labels the nuclei. Scale bar: 100 μ m. B) Co-immunoprecipitation of Pyk2 and tau in RAB fractions of brain lysates obtained from WT and pR5 mice reveals weak interaction of endogenous Pyk2 and tau in vivo . Note that cleaved forms of endogenous Pyk2 were detected in the input lysate. IP, immunoprecipitation. Three independent experiments were performed. C) Endogenous Pyk2 is present in the crude synaptosomal fraction, together with tau. WT brain tissue was processed to obtain fractions including total homogenate (Hm), supernatant S1, pellet P1 and pellet P2, followed by immunoblotting with the indicated antibodies. D) Pyk2-V5-transfected hippocampal neurons (DIV16) stained with Pyk2 and V5, revealing strong localization of Pyk2 in dendritic spines. Scale bar: 50 μ m. E) WT hippocampal neurons (DIV16) stained with PSD95 (postsynaptic marker) and MAP2. Colocalization of endogenous Pyk2 and PSD95 shown in spines of a WT neuron indicated by arrowheads. Scale bar: 50 μ m.

Article Snippet: Pyk2-V5 and Pyk2-GFP vectors were generated by cloning the Pyk2 cDNA into pcDNA6.2/V5-DEST and pcDNA6.2/N-EmGFP-DEST vectors (Gateway cloning, Thermo Fisher Invitrogen).

Techniques: Immunofluorescence, Staining, In Vitro, Cell Culture, Immunoprecipitation, In Vivo, Western Blot, Transfection, Marker

Journal: Journal of Alzheimer's Disease

Article Title: Pyk2 is a Novel Tau Tyrosine Kinase that is Regulated by the Tyrosine Kinase Fyn

doi: 10.3233/JAD-180054

Figure Lengend Snippet: Pyk2 phosphorylates tau in HEK293T cells. A) Representative immunoblots of lysates from HEK293T cells transfected with V5-tagged tau and GFP-tagged Pyk2. Validation of the Y402F mutant form of Pyk2 by immunoblotting using a phospho-Y402-Pyk2 specific antibody. Sh, short exposure; lo, long exposure. Asterisk denotes the endogenous expression of Pyk2 in HEK cells using a longer exposure of the immunoblot in the absence of Pyk2-GFP overexpression. B) Quantification of immunoblots in (A) showing relative tau and pY18-tau levels. Mean±s.e.m, n = 6 per group, one-way ANOVA, Dunnett’s multiple comparisons test, ** p < 0.01, **** p < 0.0001. C) Representative immunofluorescence staining of HEK293T cells transfected with V5-tagged tau and GFP-tagged Pyk2 indicating a strong positive correlation of pY18-tau immunoreactivity and expression of the active form of Pyk2. Scale bar: 50 μ m. D) Immunoblots of lysates from HEK293T cells co-transfected with V5-tagged tau and PTK2B or control siRNA. E) Quantification of immunoblots in (D). Mean±s.e.m, n = 3 per group, two-tailed t test, * p < 0.05, *** p < 0.001.

Article Snippet: Pyk2-V5 and Pyk2-GFP vectors were generated by cloning the Pyk2 cDNA into pcDNA6.2/V5-DEST and pcDNA6.2/N-EmGFP-DEST vectors (Gateway cloning, Thermo Fisher Invitrogen).

Techniques: Western Blot, Transfection, Mutagenesis, Expressing, Over Expression, Immunofluorescence, Staining, Two Tailed Test

Journal: Journal of Alzheimer's Disease

Article Title: Pyk2 is a Novel Tau Tyrosine Kinase that is Regulated by the Tyrosine Kinase Fyn

doi: 10.3233/JAD-180054

Figure Lengend Snippet: Pyk2 phosphorylates tau in vitro. A) Coomassie staining and immunoblots of purified recombinant His-tagged Tau-V5 and Pyk2-V5 proteins. The full-length forms of the recombinant proteins are indicated at the expected molecular weight. Carboxy-terminally cleaved Pyk2 was detected using both anti-V5 and anti-C-terminal Pyk2 antibodies. N-terminal and C-terminal Pyk2 antibodies were raised against an amino- and carboxy-terminal epitope of human Pyk2, respectively. B) Immunoblots of pY18-tau after incubation of recombinant tau with or without recombinant Pyk2 in the in vitro phosphorylation reaction. C) Immunoblots of pY18-tau after incubation of recombinant tau and Pyk2 with or without ATP in the in vitro phosphorylation reaction. D) Quantification of immunoblots in (C). Mean±s.e.m, n = 3 per group, two-tailed t test, *** p < 0.001. E) Representative immunoblots of a time course assay of Pyk2-catalyzed tau phosphorylation in vitro . Reactions were carried out at 30°C in the presence of 0.5 μ M recombinant Pyk2 and 1.9 μ M recombinant tau. F) Normalized pY18-tau levels in (E) were plotted as a function of incubation time, with fold-changes relative to time point ‘0 s’. The curve was generated by nonlinear regression. Three independent experiments were performed.

Article Snippet: Pyk2-V5 and Pyk2-GFP vectors were generated by cloning the Pyk2 cDNA into pcDNA6.2/V5-DEST and pcDNA6.2/N-EmGFP-DEST vectors (Gateway cloning, Thermo Fisher Invitrogen).

Techniques: In Vitro, Staining, Western Blot, Purification, Recombinant, Molecular Weight, Incubation, Two Tailed Test, Generated

Journal: Journal of Alzheimer's Disease

Article Title: Pyk2 is a Novel Tau Tyrosine Kinase that is Regulated by the Tyrosine Kinase Fyn

doi: 10.3233/JAD-180054

Figure Lengend Snippet: Pyk2 interacts with the tau tyrosine kinase Fyn. A) Immunoblots of cell lysates from HEK293T transfected with V5-tagged tau and Myc-tagged Fyn, indicating that Fyn robustly phosphorylates tau at Tyr18. Sh, short exposure; lo, long exposure. B) Quantification of immunoblots in (A) indicating elevation of pY18-tau levels by Fyn overexpression. Mean±s.e.m, n = 4 per group, two-tailed t test, **** p < 0.0001. C) Representative fluorescence image of HEK293T cells overexpressing Fyn-RFP and Pyk2-GFP. DAPI labels the nuclei. Scale bar: 10 μ m. D) Immunoprecipitation of Pyk2 and Fyn expressed in HEK293T cells reveals their interaction. E) Immunoprecipitation of Pyk2 and truncated forms of Fyn (Fyn Δ SH2-GFP, SH2 domain-deleted Fyn; Fyn Δ SH3-GFP, and SH3 domain-deleted Fyn) reveals the role of SH2 domain in the interaction. F) Immunoprecipitation of Fyn together with Pyk2 or a phosphorylation-deficient (inactive) Y402F mutant form of Pyk2. G) Quantification of immunoblots (F) indicating a reduced interaction between Fyn and inactive Pyk2. Mean±s.e.m, n = 4 per group, two-tailed t test, *** p < 0.001.

Article Snippet: Pyk2-V5 and Pyk2-GFP vectors were generated by cloning the Pyk2 cDNA into pcDNA6.2/V5-DEST and pcDNA6.2/N-EmGFP-DEST vectors (Gateway cloning, Thermo Fisher Invitrogen).

Techniques: Western Blot, Transfection, Over Expression, Two Tailed Test, Fluorescence, Immunoprecipitation, Mutagenesis

Journal: Journal of Alzheimer's Disease

Article Title: Pyk2 is a Novel Tau Tyrosine Kinase that is Regulated by the Tyrosine Kinase Fyn

doi: 10.3233/JAD-180054

Figure Lengend Snippet: Pyk2 is activated by Fyn in vivo. A) Immunoblots of lysates obtained from Pyk2-V5- and Fyn-Myc-transfected HEK293T cells. Mock, transfection with Lipofectamine only. Sh, short exposure; lo, long exposure. B) Quantification of immunoblots in (A) suggests that increased activity of Pyk2 is mediated by Fyn. Mean±s.e.m, n = 3 per group, two-tailed t test, n.s., not significant; ** p < 0.01; *** p < 0.001. C) Immunoblots of HEK293T cell lysates demonstrating that Pyk2 activation is Fyn activity-dependent. FynCA-Myc, constitutively active mutant Fyn (Y531F); FynKD-Myc, kinase-dead mutant Fyn (K299A). # denotes endogenous Fyn. D) Immunoblots of whole brain lysates obtained from 3-week-old Fyn CA transgenic mice and littermate controls. FynCA Tg, transgenic mice neuronally overexpress a constitutively active mutant Fyn (Y531F). E) Quantification of immunoblots in (D) suggests that the increased activity of Pyk2 is mediated by Fyn. Mean±s.e.m, n = 4 per group, two-tailed t test, **** p < 0.0001. F) Immunoblots of whole brain lysates obtained from 6-month-old Fyn knockout (FynKO) mice and WT controls. G) Quantification of immunoblots in (F). Mean±s.e.m, n = 4 per group, two-tailed t test, * p < 0.05.

Article Snippet: Pyk2-V5 and Pyk2-GFP vectors were generated by cloning the Pyk2 cDNA into pcDNA6.2/V5-DEST and pcDNA6.2/N-EmGFP-DEST vectors (Gateway cloning, Thermo Fisher Invitrogen).

Techniques: In Vivo, Western Blot, Transfection, Activity Assay, Two Tailed Test, Activation Assay, Mutagenesis, Transgenic Assay, Knock-Out