| Summary: | 碩士 === 國立陽明大學 === 生物藥學研究所 === 88 === rPICK1 binds to activated protein kinase C and is postulated to act as an intracellular receptor (or targeting subunit) of PKC which may specify location, catalytic, and regulatory properties of protein kinase C （PKC）. rPICK1 interacts with the mitogen-stimulated immediate-early gene TIS21 in the yeast two-hybrid system. We showed, in this study, that rPICK1 also associated with TIS21 in NIH 3T3 cells by co-immunoprecipitation. To investigate sequences responsible for interactions with rPICK1, the deleted mutants of TIS21 were constructed and analyzed in the yeast two-hybrid system. The N-terminal 19 amino acid residues were crucial for wild type TIS21 to interact with rPICK1, however, the C-terminal 36 amino acids were not required for the interaction. Similar results were observed with two other TIS21 interacting proteins, PRMT1 and 4A. Interestingly, the H49-L122 fragment of TIS21 alone showed a specific interaction with rPICK1. This region is likely masked in wild type TIS21. To investigate significance of the interaction between TIS21 and rPICK1, we performed in vitro kinase assay. We showed that TIS21 was phosphorylated by PKC and the phosphorylation was decreased by about 40% after preincubated with rPICK1. Phosphorylation of histone by protein kinase C was not affected by rPICk1.
Recombinant rPICK1 were purified and used to immunize rabbits. The antisera obtained were used to study the distribution of rPICK1 in NIH 3T3 cells. The endogenous rPICK1 was localized in cytoplasm with a punctate pattern. When stimulated with serum, rPICK1 translocated from cytoplasm to the perinuclear region. This change in rPICK1 localization started as early as 5 min after stimulation. At 90 min after serum stimulation the majority of the cells displayed the perinuclear pattern of rPICK1. When overexpressed, the PDZ domain-deleted Flag-rPICK1 was mainly concentrated in one or two regions around the nucleus whereas the wild type Flag-rPICK1 was found in the cytoplasm. This suggests that the PDZ domain of rPICK1 may be involved in determing its intracellular localization.
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