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 thereby activating, diverse signaling proteins. We have previously described the purification of a rat phosphatidylinositol 3-phosphate [PtdIns(3)P] 3-phosphatase, comprising a heterodimer of a 78-kDa adapter subunit in complex with a 65-kDa catalytic subunit. Here, we have cloned and characterized the cDNA encoding the human 3-phosphatase adapter subunit (3-PAP). Sequence alignment showed that 3-PAP shares significant sequence similarity with the protein and lipid 3-phosphatase myotubularin, and with several other members of the myotubularin gene family including SET-binding factor 1. However, unlike myotubularin, 3-PAP does not contain a consensus HCX(5)R catalytic motif. The 3-PAP sequence contains several motifs that predict interaction with proteins containing Src homology-2 (SH2) domains, phosphotyrosine-binding (PTB) domains, members of the 14-3-3 family, as well as proteins with SET domains. 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 immunoprecipitates isolated from platelet cytosol hydrolyzed the D3-phosphate from PtdIns(3)P and PtdIns 3,4-bisphosphate [PtdIns(3,4)P(2)]. However, insect cell-expressed 3-PAP recombinant protein was catalytically inactive, confirming our prior prediction that this polypeptide represents an adapter subunit.
Nandurkar
H H
HH
Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia. harshal.nandurkar@med.monash.edu.au
Caldwell
K K
KK
Whisstock
J C
JC
Layton
M J
MJ
Gaudet
E A
EA
Norris
F A
FA
Majerus
P W
PW
Mitchell
C A
CA
D000595:Amino Acid Sequence; D000818:Animals; D020134:Catalytic Domain; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D010716:Phosphatidylinositols / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D010802:Phylogeny; D011499:Protein Processing, Post-Translational; D021122:Protein Subunits; D017027:Protein Tyrosine Phosphatases / Q000737:chemistry*; D054558:Protein Tyrosine Phosphatases, Non-Receptor; D011506:Proteins; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015290:Second Messenger Systems; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D018076:DNA, Complementary; C489234:MTMR12 protein, human; D010716:Phosphatidylinositols; D021122:Protein Subunits; D011506:Proteins; D011993:Recombinant Fusion Proteins; D010744:Phosphoric Monoester Hydrolases; D017027:Protein Tyrosine Phosphatases; D054558:Protein Tyrosine Phosphatases, Non-Receptor; C099588:myotubularin; C061816:phosphatidylinositol-3-phosphatase