Domain organization of p130, PLC-related catalytically inactive protein, and structural basis for the lack of enzyme activity

The 130-kDa protein (p130) was isolated as a novel inositol 1,4,5-trisphosp hate [Ins(1,4,5)P-3]-binding protein similar to phospholipase C-delta 1 (PL C-delta 1), but lacking catalytic activity [Kanematsu, T., Takeya, H., Wata nabe, Y., Ozaki, S., Yoshida, M., Koga, T., Iwanaga, S. & Hirata, M. (1992) J. Biol. Chem. 267, 6518-6525; Kanematsu, T., Misumi, Y., Watanabe, Y., Oz aki, S., Koga, T, Iwanaga, S., Ikehara, Y. & Hirata, M. (1996) Biochem. J. 313, 319-325]. To test experimentally the domain organization of p130 and s tructural basis for lack of PLC activity, we subjected p130 to limited prot eolysis and also constructed a number of chimeras with PLC-delta 1. Trypsin treatment of p130 produced four major polypeptides with molecular masses o f 86 kDa, 55 kDa, 33 kDa and 25 kDa. Two polypeptides of 86 M)a and 55 kDa started at Lys93 and were calculated to end at Arg851 and Arg568, respectiv ely. Using the same approach, it has been found that the polypeptides of 33 kDa and 25 kDa are likely to correspond to regions between Val569 and Arg8 51 and Lys869 and Leu1096, respectively. All the proteolytic sites were in interconnecting regions between the predicted domains, therefore supporting domain organization based on sequence similarity to PLC-delta 1 and demons trating that all domains of p130, including the unique region at the C-term inus, are stable, tightly folded structures. p130 truncated at either or bo th the N-terminus (94 amino acids) and C-terminus (851-1096 amino acids) ex pressed in COS-1 cells showed no catalytic activity, indicating that p130 h as intrinsically no PLC activity. A number of chimeric molecules between p1 30 and PLC-delta 1 were constructed and assayed for PLC activity. It was sh own that structural differences in interdomain interactions exist between t he two proteins, as only some domains of p130 could replace the correspondi ng structures in PLC-delta 1 to form a functional enzyme. These results sug gest that p130 and the related proteins could represent a new protein famil y that may play some distinct role in cells due to the capability of bindin g Ins(1,4,5)P-3 but the lack of catalytic activity.