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

Takashi Kanematsu, Kenji Yoshimura, Kiyoshi Hidaka, Hiroshi Takeuchi, Matilda Katan, Masato Hirata

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Abstract

The 130-kDa protein (p130) was isolated as a novel inositol 1,4,5- trisphosphate [Ins(1,4,5)P3)-binding protein similar to phospholipase C-δ1 (PLC-δ1), but lacking catalytic activity [Kanematsu, T., Takeya, H., Watanabe, Y., Ozaki, S., Yoshida, M., Koga, T., Iwanaga, S. and Hirata, M. (1992) J. Biol. Chem. 267, 6518-6525, Kanematsu, T., Misumi, Y., Watanabe, Y., Ozaki, S., Koga, T., Iwanaga, S., Ikehara, Y. and Hirata, M. (1996) Biochem, J. 313, 319-3251. To test experimentally the domain organization of p130 and structural basis for lack of PLC activity, we subjected p130 to limited proteolysis and also constructed a number of chimeras with PLC-δ1. Trypsin treatment of p130 produced four major polypeptides with molecular misses of 86 kDa, 55 kDa, 33 kDa and 25 kDa. Two polypeptides of 86 kDa and 55 kDa started at Lys93 and were CalCulated to end at Arg851 and Arg568, respectively. 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 Arg851 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- δ1 and demonstrating that all domains of p130, including the unique region at the C-terminus, are stable, tightly folded structures. p130 truncated at either or both the N-terminus (94 amino acids) and C-terminus (851-1096 amino acids) expressed in COS-1 cells showed no catalytic activity, indicating that p130 has intrinsically no PLC activity. A number of chimeric molecules between p130 and PLC-δ1 were constructed and assayed for PLC activity. It was shown that structural differences in interdomain interactions exist between the two proteins, as only some domains of p130 could replace the corresponding structures in PLC-δ1 to form a functional enzyme. These results suggest that p130 and the related proteins could represent a new protein family that may play some distinct role in cells due to the capability of binding Ins(1,4,5)P3 but the lack of catalytic activity.

Original languageEnglish
Pages (from-to)2731-2737
Number of pages7
JournalEuropean Journal of Biochemistry
Volume267
Issue number9
DOIs
Publication statusPublished - May 30 2000

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Enzyme activity
Type C Phospholipases
Programmable logic controllers
Catalyst activity
Enzymes
Peptides
Proteins
Proteolysis
Amino Acids
Inositol 1,4,5-Trisphosphate
COS Cells
Trypsin
Carrier Proteins
Molecules

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Domain organization of p130, PLC-related catalytically inactive protein, and structural basis for the lack of enzyme activity. / Kanematsu, Takashi; Yoshimura, Kenji; Hidaka, Kiyoshi; Takeuchi, Hiroshi; Katan, Matilda; Hirata, Masato.

In: European Journal of Biochemistry, Vol. 267, No. 9, 30.05.2000, p. 2731-2737.

Research output: Contribution to journalArticle

Kanematsu, Takashi ; Yoshimura, Kenji ; Hidaka, Kiyoshi ; Takeuchi, Hiroshi ; Katan, Matilda ; Hirata, Masato. / Domain organization of p130, PLC-related catalytically inactive protein, and structural basis for the lack of enzyme activity. In: European Journal of Biochemistry. 2000 ; Vol. 267, No. 9. pp. 2731-2737.
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AU - Katan, Matilda

AU - Hirata, Masato

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