Functional Analysis Of Human Cdc2-Related Kinase HPFTAIRE1 And Its Interacting Proteins

Degenerate PCR was performed for screening novel Cdc2-related proteinkinases. Using an EST fragment isolated from degenerate PCR as probe to screenHela cell cDNA library, one gene encoding novel Cdc2-related kinases was isolatedand designated hPFTAIRE1. The coding region of hPFTAIRE1 gene is 1410bp,which encodes a 469 amino acid protein. The entire hPFTAIRE1 amino acidsequence has the highest homology with mouse PFTAIRE1 (95%). The centraldomain (40-440 aa) is 53% identical to Cdc2 and contains the conserved ATP bindingsite and Ser/Thr phosphorylation catalytic site. The region of N-terminal 140 aminoacids is specific to hPFTAIRE1 and contains two deduced nuclear localization signals.The GFP-hPFTAIRE1 fusion protein exhibits cytoplasmid distribution in Hela cells.hPFTAIRE1 is highly expressed in human brain, testis, ovary, heart, kidney andpancreas. To search the possible regulators, substrates and the cytoplasmicdistribution mechanism of hPFTAIRE1, we screened a yeast LexA two-hybrid libraryof adult human brain with full-length hPFTAIRE1 as a bait. In this screening, weobtained 7 proteins which interact with the hPFTAIRE1, including 14-3-3β, 14-3-3θ,14-3-3η, 14-3-3ε, KIAA0202, PLZF and Cyclin Y.14-3-3 proteins are a family of highly conserved acidic proteins, which areespecially abundant in brain cells and other CNS cells. We found a putative 14-3-3binding consensus motif (RHSSPSS) in the hPFTAIRE1, which overlapped with itssecond NLS. Deletion of the RHSSPSS motif or substitution of Ser119 with Ala inthe conserved binding motif abolished the specific interaction between thehPFTAIRE1 and the 14-3-3 proteins. The mutant S120A hPFTAIRE1 also showed aweak interaction to the 14-3-3 proteins. The results suggested that the Ser119 is crucialfor the interaction between hPFTAIRE1 and the 14-3-3 proteins. All the hPFTAIRE1mutants distributed in cytoplasm of Hela cells and human neuroblastoma cells(SH-SY5Y) when fused to the C-terminus of a green fluorescent protein (GFP),indicating that binding with the 14-3-3 proteins does not contribute to the subcellularlocalization of the hPFTAIRE1, although the binding may be involved in its signalingregulation.Promyelocytic Leukemia Zinc Finger (PLZF) is also one of the sevenhPFTAIRE1 interacting proteins. PLZF encodes a transcription factor belonging to thePOZ/ BTB domain and Krüppel zinc finger (POK) family. The highly conservedPOZ/ BTB domain plays a critical role in protein-protein interaction. The interactionbetween the PLZF and the hPFTAIRE1 was confirmed by β-galactosidase assay andLeu growth activity. We deleted the POZ/ BTB domain and Krüppel zinc fingerdomain respectively, and check the interaction between hPFTAIRE1 and truncatedPLZFs by using liquid β-galactosidase activity assay. A weak interaction was detectedbetween hPFTAIRE1 and PLZF. We also checked the interaction between PLZF andanother Cdc2-related kinase PCTAIRE1. A similar result was observed. Theinteraction between the PLZF and the hPFTAIRE1 or PCTAIRE1 was confirmed byCo-immunoprecipitation assay in yeast system. PLZF is a phosphoprotein and plays amultiple role during cell growth. Our results suggest that the hPFTAIRE1 andPCTAIRE1 may play important roles in functional regulation of the PLZF.In the two-hybrid screening we also obtained a novel gene, Cyclin Y. The codingregion of the Cyclin Y is 1026 bp, which encodes a 341 amino acid protein containinga conserved Cyclin-box domain. The CyclinY contains a conserved 5 helices, thoughthe overall sequence similarity between Cyclin Y and other cyclins is very low. Threetranscripts were detected in the Northern blotting analysis. The 4.2 kb and 4.0 kbtranscripts were constitutively expressed in 16 human tissues at a relatively low level.Interestingly, the 2.0 kb transcript of Cyclin Y was highly expressed in testis, heart,skeleton muscle and rarely detected in the other tissues. The GFP-CyclinY fusionprotein was ectopically expressed in Hela cells and observed under a florescencemicroscope. The microscopy results indicated that the Cyclin Y distributes all over thecells, including cytoplasm and nucleus. To search its possible partners, we screened ayeast LexA two-hybrid library of adult human brain with full-length Cyclin Y as a baitand obtained another Cdc2 related protein kinase PCTAIRE1 which interacts with theCyclin Y, besides hPFTAIRE1. The interactions between them were confirmed inyeast system.