Characterization of an adapter subunit to a phosphatidylinositol (3)P 3-phosphatase: Identification of a myotubularin-related protein lacking catalytic activity

The D3-phosphoinositides act as second messengers by recruiting, and thereb y activating, diverse signaling proteins. We have previously described the purification of a rat phosphatidylinositol 3-phosphate [PtdIns(3)P] 3-phosp hatase, comprising a heterodimer of a 78-kDa adapter subunit in complex wit h a 65-kDa catalytic subunit. Here, we have cloned and characterized the cD NA encoding the human 3-phosphatase adapter subunit (3-PAP). Sequence align ment showed that 3-PAP shares significant sequence similarity with the prot ein and lipid 3-phosphatase myotubularin, and with several other members of the myotubularin gene family including SET-binding factor 1. However, unli ke myotubularin, 3-PAP does not contain a consensus HCX5R catalytic motif. The 3-PAP sequence contains several motifs that predict interaction with pr oteins containing Src homology-2 (SH2) domains, phosphotyrosine-binding (PT B) domains, members of the 14-3-3 family, as well as proteins with SET doma ins. Northern blot analysis identified two transcripts (5.5 kb and 2.5 kb) with highest abundance in human liver, kidney, lung, and placenta. 3-PAP im munoprecipitates isolated from platelet cytosol hydrolyzed the D3-phosphate from PtdIns(3)P and PtdIns 3,4-bisphosphate [PtdIns(3,4)P-2]. However, ins ect cell-expressed 3-PAP recombinant protein was catalytically inactive, co nfirming our prior prediction that this polypeptide represents an adapter s ubunit.