Synaptojanin Is the Major Constitutively Active Phosphatidylinositol-3,4,5-trisphosphate 5-Phosphatase in Rodent Brain

doi:10.1074/jbc.272.15.9625

Synaptojanin Is the Major Constitutively Active Phosphatidylinositol-3,4,5-trisphosphate 5-Phosphatase in Rodent Brain*

From the ^‡ Protein Phosphorylation Laboratory, Imperial Cancer Research Fund, 44 Lincoln's Inn Fields, London WC2A 3PX
^§ Yamanouchi Research Institute, Littlemore Park, Oxford, OX4 4SX
^¶ Ludwig Institute for Cancer Research, 91 Riding House Street
London, W1P 8BT, and ^∥ Department of Biochemistry and Molecular Biology, University College, Gower Street, London WC1E 6BT, United Kingdom

** To whom correspondence should be addressed:
Protein Phosphorylation Laboratory, Imperial Cancer Research Fund, P. O. Box 123, Lincoln's Inn Fields, London WC2A 3PX, UK.
Tel.: 44-171-269-3460; Fax: 44-171-269-3092.

Abstract

The major constitutive phosphatidylinositol-3,4,5-P₃ (PtdIns) 5-phosphatase activity was purified and subjected to peptide sequence analysis providing extensive amino acid sequence which was subsequently used for cloning the cDNA. Peptide and cDNA sequences revealed that the purified PtdIns(3,4,5)P₃ 5-phosphatase was identical to a splice variant of a recently cloned inositol polyphosphate 5-phosphatase termed synaptojanin. Since synaptojanin is not known to possess PtdIns(3,4,5)P₃ 5-phosphatase activity, we verified that the purified PtdIns(3,4,5)P₃ 5-phosphatase activity and synaptojanin are identical by Western blot using specific antibodies raised against synaptojanin sequences. Immunoprecipitation from crude lysates of rat brain tissue showed that synaptojanin accounts for the major part of the active PtdIns(3,4,5)P₃ 5-phosphatase activity. It is also shown that the protein is localized to the soluble fraction. Expression of a truncated recombinant protein demonstrates that the conserved 5-phosphatase region of the synaptojanin gene expresses PtdIns(3,4,5)P₃ 5-phosphatase activity. However, immunological analysis demonstrates that the PtdIns(3,4,5)P₃ 5-phosphatase activity expressed from the synaptojanin gene in brain is due to a particular splice variant which contains a 16-amino acid insert as shown by immunoprecipitation using a specific antibody raised against this particular splice variant.

Footnotes

↵* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

PtdIns

phosphatidylinositol

PIP₃

phosphatidylinositol 3,4,5-trisphosphate

PCR

polymerase chain reaction

PVDF

polyvinylidene difluoride.
- Received January 14, 1997.