Arsenic-Induced Activation of the Homeodomain-Interacting Protein Kinase 2 (HIPK2) to cAMP-Response Element Binding Protein (CREB) Axis

Abstract

CREB (cAMP-response element binding protein) plays key transcriptional roles in cell metabolism, proliferation, and survival. Ser133 phosphorylation by protein kinase A (PKA) is a well-characterized CREB activation mechanism. HIPK2 (homeodomain interacting protein kinase 2), a nuclear serine/threonine kinase, activates CREB through Ser271 phosphorylation; however, the regulatory mechanism remains uncharacterized. Transfection of CREB in HEK293 cells together with the kinase demonstrated that HIPK2 phosphorylated CREB at Ser271 but not Ser133, likewise PKA phosphorylated CREB at Ser133 but not Ser271, suggesting two distinct CREB regulatory mechanisms by HIPK2 and PKA. In vitro kinase assay revealed that HIPK2 as well as HIPK1 and HIPK3 directly phosphorylated CREB. Cells exposed to 10 μM sodium arsenite increased the stability of HIPK1 and HIPK2 proteins leading to CREB activation via Ser271 phosphorylation. Phospho-Ser271 CREB showed facilitated interaction with the TFIID subunit coactivator TAF4 assessed by immunoprecipitation. Furthermore, a focused gene array between cells transfected with CREB alone and CREB plus HIPK2 over empty vector-transfected control displayed 14- and 32-fold upregulation of CCNA1 (cyclin A1), respectively, while no upregulation by HIPK2 alone. These results suggest that the HIPK2-phospho-Ser271 CREB axis is a new arsenic-responsive CREB activation mechanism in parallel with the PKA-phospho-Ser133 CREB axis.

Characterization of an RNA binding protein interactome reveals the targetable post-transcriptional landscape of MYC-amplified medulloblastoma

“…Our stringent and integrative analysis across multiple experimental platforms identi ed HIPK1 as the top MSI1-downstream G3 MB targeting (i.e., not MSI1 bound in NSC) candidate gene for potential therapeutic drug discovery (Figure 4a, Supplementary Figure 9a). Functionally, HIPK1 has been observed to phosphorylate the cAMP response element binding (CREB) protein 101 , whose microdeletion is associated with Rubinstein-Taybi syndrome, a known G3 MB associated disease 102,103 indicating clinical relevance of HIPK1 targeting in G3 MB. Interestingly, in our platform, HIPK2 was MSI1-bound in both G3 MB and NSC stem cells whereas differential binding of HIPK1 was identi ed only in G3 MB.…”

Transmembrane protein western blotting: Impact of sample preparation on detection of SLC11A2 (DMT1) and SLC40A1 (ferroportin)

“…Klenow fragment, and cloned into SmaI site of pCMV and pCMVFlag vectors. These plasmid DNAs, verified by DNA sequencing (Eurofins), were transiently transfected into HEK293 cells by electroporation (X-Cell, Bio-Rad) as described previously [ 36 ].…”

“…Klenow fragment, and cloned into SmaI site of pCMV and pCMVFlag vectors. These plasmid DNAs, verified by DNA sequencing (Eurofins), were transiently transfected into HEK293 cells by electroporation (X-Cell, Bio-Rad) as described previously [36]. Human Fpn1 siRNA was purchased from Sigma-Aldrich (GAUGGAACUUGGUAUCCAU[dT] [dT], and AUGGAUACCAAGUUCCAUC[dT][dT]) and 5ul of 20uM solution was transfected into Caco-2 cells (grown in 2ml media/35 mm plate) with 200ul of Opti-MEM containing 6ul of Lipofectamine RNAiMax (Invitrogen) for 18hr, followed by treatment with 250 and 500uM FAC for additional 8hr in the presence of siRNA prior to harvest.…”

CircRNA-016901 silencing attenuates irradiation-induced injury in bone mesenchymal stem cells via regulating the miR-1249-5p/HIPK2 axis

“…Homeodomain-interacting protein kinase (HIPK) is a serine/threonine protein kinase located in the nucleus and the HIPK subfamily includes HIPK1, HIPK2 and HIPK3 (48). As a transcription factor, HIPK2 regulates cell differentiation, proliferation, angiogenesis and apoptosis and is associated with tumor development and progression (49)(50)(51). A recent study has reported that HIPK2 is a tumor suppressor gene that affects the biological characteristics of various tumors, including esophageal squamous cell carcinoma (52).…”