SYNAPTOJANIN IS THE MAJOR CONSTITUTIVELY ACTIVE PHOSPHATIDYLINOSITOL-3,4,5-TRISPHOSPHATE 5-PHOSPHATASE IN RODENT BRAIN

The major constitutive phosphatidylinositol-3,4,5-P-3 (PtdIns) 5-phosp hatase activity was purified and subjected to peptide sequence analysi s providing extensive amino acid sequence which was subsequently used for cloning the cDNA. Peptide and cDNA sequences revealed that the pur ified PtdIns(3,4,5)P-3 5-phosphatase was identical to a splice variant of a recently cloned inositol polyphosphate 5-phosphatase termed syna ptojanin. Since synaptojanin is not known to possess PtdIns(3,4,5)P-3 5-phosphatase activity, we verified that the purified PtdIns(3,4,5)P-3 5-phosphatase activity and synaptojanin are identical by Western blot using specific antibodies raised against synaptojanin sequences, Immu noprecipitation from crude lysates of rat brain tissue showed that syn aptojanin accounts for the major part of the active PtdIns(3,4,5)P-3 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 synaptoja nin gene expresses PtdIns(3,4,5)P-3 5-phosphatase activity, However, i mmunological analysis demonstrates that the PtdIns(3,4,5)P-3 5-phospha tase activity expressed from the synaptojanin gene in brain is due to a particular splice variant which contains a 16-amino acid insert as s hown by immunoprecipitation using a specific antibody raised against t his particular splice variant.