Journal: Cancers

Article Title: NEK1 Phosphorylation of YAP Promotes Its Stabilization and Transcriptional Output

doi: 10.3390/cancers12123666

Figure Lengend Snippet: ( A ) The expression of yes-associated protein (YAP) was regulated by never in mitosis gene A (NIMA)-related kinases (NEK) activity and its upstream kinase tousled-like kinase (TLK). Overexpression of wt-NEK1 resulted in elevated YAP expression and conversely in its degradation in LNCaP cells overexpressing the dominant negative mutant NEK1-T141A. Thioridazine (THD) led to degradation of YAP in parental LNCaP cells, even after treatment with bicalutamide (BIC), which led to overexpression of TLK1B. ( B ) YAP interacted with NEK1 and was enriched upon co-immunoprecipitation. TLK1 inhibition with 10 µM THD did not affect NEK1 interaction with YAP, and thus the state of NEK1 kinase activity did not affect YAP binding. ( C ) The expression of YAP was decreased in NT1 cells treated with two different inhibitors of TLK (THD and J54), with a corresponding increase in CL-YAP products. ( D ) Expression of several typical YAP target genes in LNCaP cells treated with THD.

Article Snippet: The following antibodies were used in this study: mouse anti-YAP (Santa Cruz Biotechnology, SCBT, Dallas, TX, USA, cat# sc101199), rabbit anti-phospho-YAP (Cell Signaling Technology, CST, Dallas, TX, USA, cat# 13008), mouse anti-NEK1 (SCBT, cat# sc 398813, Dallas, TX, USA), rabbit anti-phospho-NEK1 pT141 (lab-generated), rabbit anti-phospho-tyrosine (CST, cat# 8954S, Dallas, TX, USA), HRP-conjugated anti-β-tubulin (SCBT, Dallas, TX, USA, cat# sc-23949), mouse IgG (SCBT, Dallas, TX, USA, cat# sc-2025), and rabbit anti-actin (Abcam, Cambridge, MA, USA, cat# ab1801).

Techniques: Expressing, Activity Assay, Over Expression, Dominant Negative Mutation, Immunoprecipitation, Inhibition, Binding Assay

Journal: Cancers

Article Title: NEK1 Phosphorylation of YAP Promotes Its Stabilization and Transcriptional Output

doi: 10.3390/cancers12123666

Figure Lengend Snippet: ( A ) CRISPR/Cas9-mediated loss of NEK1 resulted in reduced levels of YAP protein, possibly due to instabilization (EV = empty vector). ( B ) Expression of several typical YAP target genes is reduced in NEK1 KO clones. GAPDH mRNA was used as an internal control. ( C ) Treatment of LNCaP cells with THD, a specific inhibitor of TLKs, resulted in reduced YAP protein level and conversely in its S127 hyperphosphorylation. ( D ) Reduction of TLK1 expression via (short hairpin) shRNA transfection led to loss of pNEK1-T141.

Article Snippet: The following antibodies were used in this study: mouse anti-YAP (Santa Cruz Biotechnology, SCBT, Dallas, TX, USA, cat# sc101199), rabbit anti-phospho-YAP (Cell Signaling Technology, CST, Dallas, TX, USA, cat# 13008), mouse anti-NEK1 (SCBT, cat# sc 398813, Dallas, TX, USA), rabbit anti-phospho-NEK1 pT141 (lab-generated), rabbit anti-phospho-tyrosine (CST, cat# 8954S, Dallas, TX, USA), HRP-conjugated anti-β-tubulin (SCBT, Dallas, TX, USA, cat# sc-23949), mouse IgG (SCBT, Dallas, TX, USA, cat# sc-2025), and rabbit anti-actin (Abcam, Cambridge, MA, USA, cat# ab1801).

Techniques: CRISPR, Plasmid Preparation, Expressing, Clone Assay, Control, shRNA, Transfection

Journal: Cancers

Article Title: NEK1 Phosphorylation of YAP Promotes Its Stabilization and Transcriptional Output

doi: 10.3390/cancers12123666

Figure Lengend Snippet: ( A ) Expression and purification of His-NEK1 kinase domain (NEK1∆CT). ( B ) NEK1∆CT was catalytically active and ATP/ADP conversion (kinase activity) was linear with the enzyme amount. ( C ) In vitro phosphorylation reactions of YAP using His-NEK1 and MK5 kinases in presence of [γ- 32 P] ATP. ( D ) In vitro phosphorylation of YAP using His-NEK1 and MK5 kinases for preparative isolation for MS determination of phosphopeptides. ( E ) His-NEK1 also phosphorylated YAP on Tyr, as demonstrated by immunoreactivity with pY antibody.

Article Snippet: The following antibodies were used in this study: mouse anti-YAP (Santa Cruz Biotechnology, SCBT, Dallas, TX, USA, cat# sc101199), rabbit anti-phospho-YAP (Cell Signaling Technology, CST, Dallas, TX, USA, cat# 13008), mouse anti-NEK1 (SCBT, cat# sc 398813, Dallas, TX, USA), rabbit anti-phospho-NEK1 pT141 (lab-generated), rabbit anti-phospho-tyrosine (CST, cat# 8954S, Dallas, TX, USA), HRP-conjugated anti-β-tubulin (SCBT, Dallas, TX, USA, cat# sc-23949), mouse IgG (SCBT, Dallas, TX, USA, cat# sc-2025), and rabbit anti-actin (Abcam, Cambridge, MA, USA, cat# ab1801).

Techniques: Expressing, Purification, Activity Assay, In Vitro, Phospho-proteomics, Isolation

Journal: Cancers

Article Title: NEK1 Phosphorylation of YAP Promotes Its Stabilization and Transcriptional Output

doi: 10.3390/cancers12123666

Figure Lengend Snippet: Gene expression of ( A ) NEK1 and ( B ) YAP1 of prostate adenocarcinoma (PRAD) patients on the basis of the Gleason score extracted from The Cancer Genome Atlas (TCGA) datasets using the UALCAN web tool. OncoPrint representation of the protein level alteration of ( C ) NEK1 and ( D ) YAP1 of PRAD patients by reverse phase protein array (RPPA) extracted from TCGA (firehose legacy) datasets using cBIOPORTAL online platform. Gene expression of ( E ) NEK1 and ( F ) YAP1 in head and neck squamous cell (HNSC) patients on the basis of the tumor grade extracted from TCGA datasets using the UALCAN web tool. Percentage of patients of different types of cancer with higher level of ( G ) NEK1 and ( H ) YAP1 on the basis of immunohistochemistry (IHC) staining generated using the Human Protein Atlas database. Staining intensity correlated with the color code. Deeper color represents high staining intensity. ( I ) Representative IHC images of NEK1 and YAP1 of high-grade PRAD (top panel) and metastatic HNSC samples (bottom panel). ( J ) Volcano plot of gene enrichment analysis based on NEK1 overexpression in HNSC patients extracted from TCGA (firehose legacy) datasets using cBIOPORTAL online platform. * represents p < 0.05, ** represents p < 0.005, and *** represents p < 0.0005. All comparisons were with the normal tissue.

Article Snippet: The following antibodies were used in this study: mouse anti-YAP (Santa Cruz Biotechnology, SCBT, Dallas, TX, USA, cat# sc101199), rabbit anti-phospho-YAP (Cell Signaling Technology, CST, Dallas, TX, USA, cat# 13008), mouse anti-NEK1 (SCBT, cat# sc 398813, Dallas, TX, USA), rabbit anti-phospho-NEK1 pT141 (lab-generated), rabbit anti-phospho-tyrosine (CST, cat# 8954S, Dallas, TX, USA), HRP-conjugated anti-β-tubulin (SCBT, Dallas, TX, USA, cat# sc-23949), mouse IgG (SCBT, Dallas, TX, USA, cat# sc-2025), and rabbit anti-actin (Abcam, Cambridge, MA, USA, cat# ab1801).

Techniques: Gene Expression, Protein Array, Immunohistochemistry, Generated, Staining, Over Expression

Journal: Cancers

Article Title: NEK1 Phosphorylation of YAP Promotes Its Stabilization and Transcriptional Output

doi: 10.3390/cancers12123666

Figure Lengend Snippet: Some of the YAP target genes significantly upregulated with NEK1 upregulation in head and neck squamous cell (HNSC) carcinoma (TCGA, firehose legacy) analyzed using cBIOPORTAL.

Article Snippet: The following antibodies were used in this study: mouse anti-YAP (Santa Cruz Biotechnology, SCBT, Dallas, TX, USA, cat# sc101199), rabbit anti-phospho-YAP (Cell Signaling Technology, CST, Dallas, TX, USA, cat# 13008), mouse anti-NEK1 (SCBT, cat# sc 398813, Dallas, TX, USA), rabbit anti-phospho-NEK1 pT141 (lab-generated), rabbit anti-phospho-tyrosine (CST, cat# 8954S, Dallas, TX, USA), HRP-conjugated anti-β-tubulin (SCBT, Dallas, TX, USA, cat# sc-23949), mouse IgG (SCBT, Dallas, TX, USA, cat# sc-2025), and rabbit anti-actin (Abcam, Cambridge, MA, USA, cat# ab1801).

Techniques: Expressing

Journal: Cancers

Article Title: NEK1 Phosphorylation of YAP Promotes Its Stabilization and Transcriptional Output

doi: 10.3390/cancers12123666

Figure Lengend Snippet: ( A ) The expression of yes-associated protein (YAP) was regulated by never in mitosis gene A (NIMA)-related kinases (NEK) activity and its upstream kinase tousled-like kinase (TLK). Overexpression of wt-NEK1 resulted in elevated YAP expression and conversely in its degradation in LNCaP cells overexpressing the dominant negative mutant NEK1-T141A. Thioridazine (THD) led to degradation of YAP in parental LNCaP cells, even after treatment with bicalutamide (BIC), which led to overexpression of TLK1B. ( B ) YAP interacted with NEK1 and was enriched upon co-immunoprecipitation. TLK1 inhibition with 10 µM THD did not affect NEK1 interaction with YAP, and thus the state of NEK1 kinase activity did not affect YAP binding. ( C ) The expression of YAP was decreased in NT1 cells treated with two different inhibitors of TLK (THD and J54), with a corresponding increase in CL-YAP products. ( D ) Expression of several typical YAP target genes in LNCaP cells treated with THD.

Article Snippet: A total of 50 μL of equilibrated protein A/G agarose (SCBT, Dallas, TX, USA, cat# sc-2003) was incubated with either mouse anti-NEK1 antibody or mouse IgG antibody at 4 °C for 4 h with rotation.

Techniques: Expressing, Activity Assay, Over Expression, Dominant Negative Mutation, Immunoprecipitation, Inhibition, Binding Assay

Journal: Cancers

Article Title: NEK1 Phosphorylation of YAP Promotes Its Stabilization and Transcriptional Output

doi: 10.3390/cancers12123666

Figure Lengend Snippet: ( A ) CRISPR/Cas9-mediated loss of NEK1 resulted in reduced levels of YAP protein, possibly due to instabilization (EV = empty vector). ( B ) Expression of several typical YAP target genes is reduced in NEK1 KO clones. GAPDH mRNA was used as an internal control. ( C ) Treatment of LNCaP cells with THD, a specific inhibitor of TLKs, resulted in reduced YAP protein level and conversely in its S127 hyperphosphorylation. ( D ) Reduction of TLK1 expression via (short hairpin) shRNA transfection led to loss of pNEK1-T141.

Article Snippet: A total of 50 μL of equilibrated protein A/G agarose (SCBT, Dallas, TX, USA, cat# sc-2003) was incubated with either mouse anti-NEK1 antibody or mouse IgG antibody at 4 °C for 4 h with rotation.

Techniques: CRISPR, Plasmid Preparation, Expressing, Clone Assay, Control, shRNA, Transfection

Journal: Cancers

Article Title: NEK1 Phosphorylation of YAP Promotes Its Stabilization and Transcriptional Output

doi: 10.3390/cancers12123666

Figure Lengend Snippet: ( A ) Expression and purification of His-NEK1 kinase domain (NEK1∆CT). ( B ) NEK1∆CT was catalytically active and ATP/ADP conversion (kinase activity) was linear with the enzyme amount. ( C ) In vitro phosphorylation reactions of YAP using His-NEK1 and MK5 kinases in presence of [γ- 32 P] ATP. ( D ) In vitro phosphorylation of YAP using His-NEK1 and MK5 kinases for preparative isolation for MS determination of phosphopeptides. ( E ) His-NEK1 also phosphorylated YAP on Tyr, as demonstrated by immunoreactivity with pY antibody.

Article Snippet: A total of 50 μL of equilibrated protein A/G agarose (SCBT, Dallas, TX, USA, cat# sc-2003) was incubated with either mouse anti-NEK1 antibody or mouse IgG antibody at 4 °C for 4 h with rotation.

Techniques: Expressing, Purification, Activity Assay, In Vitro, Phospho-proteomics, Isolation

Journal: Cancers

Article Title: NEK1 Phosphorylation of YAP Promotes Its Stabilization and Transcriptional Output

doi: 10.3390/cancers12123666

Figure Lengend Snippet: Gene expression of ( A ) NEK1 and ( B ) YAP1 of prostate adenocarcinoma (PRAD) patients on the basis of the Gleason score extracted from The Cancer Genome Atlas (TCGA) datasets using the UALCAN web tool. OncoPrint representation of the protein level alteration of ( C ) NEK1 and ( D ) YAP1 of PRAD patients by reverse phase protein array (RPPA) extracted from TCGA (firehose legacy) datasets using cBIOPORTAL online platform. Gene expression of ( E ) NEK1 and ( F ) YAP1 in head and neck squamous cell (HNSC) patients on the basis of the tumor grade extracted from TCGA datasets using the UALCAN web tool. Percentage of patients of different types of cancer with higher level of ( G ) NEK1 and ( H ) YAP1 on the basis of immunohistochemistry (IHC) staining generated using the Human Protein Atlas database. Staining intensity correlated with the color code. Deeper color represents high staining intensity. ( I ) Representative IHC images of NEK1 and YAP1 of high-grade PRAD (top panel) and metastatic HNSC samples (bottom panel). ( J ) Volcano plot of gene enrichment analysis based on NEK1 overexpression in HNSC patients extracted from TCGA (firehose legacy) datasets using cBIOPORTAL online platform. * represents p < 0.05, ** represents p < 0.005, and *** represents p < 0.0005. All comparisons were with the normal tissue.

Article Snippet: A total of 50 μL of equilibrated protein A/G agarose (SCBT, Dallas, TX, USA, cat# sc-2003) was incubated with either mouse anti-NEK1 antibody or mouse IgG antibody at 4 °C for 4 h with rotation.

Techniques: Gene Expression, Protein Array, Immunohistochemistry, Generated, Staining, Over Expression

Journal: Cancers

Article Title: NEK1 Phosphorylation of YAP Promotes Its Stabilization and Transcriptional Output

doi: 10.3390/cancers12123666

Figure Lengend Snippet: Some of the YAP target genes significantly upregulated with NEK1 upregulation in head and neck squamous cell (HNSC) carcinoma (TCGA, firehose legacy) analyzed using cBIOPORTAL.

Article Snippet: A total of 50 μL of equilibrated protein A/G agarose (SCBT, Dallas, TX, USA, cat# sc-2003) was incubated with either mouse anti-NEK1 antibody or mouse IgG antibody at 4 °C for 4 h with rotation.

Techniques: Expressing