Interaction of the regulatory subunit of the cAMP-dependent protein kinase with PATZ1 (ZNF278)

Abstract

The effects of cAMP in cell are predominantly mediated by the cAMP-dependent protein kinase (PKA), which is composed of two genetically distinct subunits, catalytic (C) and regulatory (R), forming a tetrameric holoenzyme R(2)C(2). The only known function for the R subunit is that of inhibiting the activity of the C subunit kinase. It has been shown that overexpression of RIalpha, but not the C subunit kinase, is associated with neoplastic transformation. In addition, it has also been demonstrated that mutation in the RIalpha, but not the C subunit is associated with increased resistance to the DNA-damaging anticancer drug cisplatin, thus suggesting that the RIalpha subunit of PKA may have functions independent of the kinase. We show here that the RIalpha subunit interacts with a BTB/POZ domain zinc-finger transcription factor, PATZ1 (ZNF278), and co-expression with RIalpha results in its sequestration in the cytoplasm. The cytoplasmic/nuclear translocation is inducible by cAMP. C-terminus deletion abolishes PATZ1 interaction with RIalpha and results in its localization in the nucleus. PATZ1 transactivates the cMyc promoter and the presence of cAMP and co-expression with RIalpha modulates its transactivation. Moreover, PATZ1 is aberrantly expressed in cancer. Taken together, our results showed a potentially novel mechanism of cAMP signaling mediated through the interaction of RIalpha with PATZ1 that is independent of the kinase activity of PKA, and the aberrant expression of PATZ1 in cancer point to its role in cell growth regulation.

Fig. 1

Interaction of RIα with PATZ1. (A) Yeast two-hybrid interaction experiment showing the association of RIα with PATZ1. (B) Schematic representation of the full length and the upstream ATGs-deletion mutant of PATZ1. Approximately 350 bp from the 5′ end of PATZ1 cDNA, containing a cluster of upstream ATGs, was deleted. (C) In vitro transcription and translation was performed with the full length PATZ1 or its upstream ATGs-deletion mutant (•PATZ1) compared to the pBluescript II SK(−) vector control. (D) The in vitro-[³⁵S]Methionine-labeled translated PATZ1 was interacted with either GST or GST-RIα immobilized on glutathione resins. (E) Schematic representation of wild-type and deletion mutants of RIα. Dimer. Dom., the N-terminal dimerization domain; Inh. Site, inhibitory site; Sites A and B are the two cAMP binding domains. (F) Top panel, interaction of PATZ1 from yeast extracts with either recombinant GST, GST-RIα, GST-RIα(Δ1-76), and GST-RIα(Δ77-380); bottom panel, bacterial lysates containing the respective expressed proteins GST, GST-RIα; GST-RIα(Δ77-380) and GST-RIα(Δ1-76).

Fig. 2

Subcellular localization of RIAZ. A, C, E, and G, light micrograph of PC3M cells (A, C) or normal fibroblasts (E, G) either transfected with GFP/PATZ1 (A, B, E, F) or cotransfected with GFP/PATZ1 and RIα (C, D, G, H). B, D, F, H, are fluorescence image of GFP/PATZ1 localization. I, fluorescence image of PC3M cells transfected with GFP alone; and J, cotransfected with GFP and RIα. K, L, localization of either GFP/PATZ1 or PATZ1/GFP in HTB-46 cells; L, transfected with RIAZ/GFP; M, cotransfected with GFP/RIAZ and RIα in the absence of 8-Br-cAMP; N, cotransfected with GFP/RIAZ and RIα in the presence of 1 mM 8-Br-cAMP. Subcellular localization of GFP/RIAZ was visualized using a Zeiss Axioskop fluorescence microscope.

Fig. 3

Interaction with RIα with PATZ1 deletion mutant. PC3M cells were transfected with (A) GFP/PATZ1; (B) GFP/PATZ1 and RIα; (C) GFP fusion of C-terminus deletion mutant of PATZ1 (GFP/Δ PATZ1); or (D) GFP/ΔPATZ1 and RIα. Subcellular localization of GFP/PATZ1 was visualized 24 hr after transfection.

Fig. 4

Expression of PATZ1 in normal human tissue, cancer cell lines, and clinical cancer specimens. (A) RNA blot analysis of PATZ1 in normal human tissues. Northern blot analysis of PATZ1 in various (C) human cancer cell lines, and a panel of (C) breast cancer cells. (D) PATZ1 expression in breast cancer specimens from 119 patients. Red bars in dendrogram showed increased PATZ1 levels, and green bar, decreased levels compared with reference to mean.