Specificity of the myotubularin family of phosphatidylinositol-3-phosphatase is determined by the PH/GRAM domain.
Myotubularins (MTM) are a large subfamily of lipid phosphatases that specifically dephosphorylate at the D3 position of phosphatidylinositol 3-phosphate (PI(3)P) in PI(3)P and PI(3,5)P2. We recently found that MTMR6 specifically inhibits the Ca2+-activated K+ channel, KCa3.1, by dephosphorylating PI(3)P. We now show that inhibition is specific for MTMR6 and other MTMs do not inhibit KCa3.1. By replacing either or both of the coiled-coil (CC) and pleckstrin homology/GRAM (PH/G) domains of MTMs that failed to inhibit KCa3.1 with the CC and PH/G domains of MTMR6, we found that chimeric MTMs containing both the MTMR6 CC and PH/G domains functioned like MTMR6 to inhibit KCa3.1 channel activity, whereas chimeric MTMs containing either domain alone did not. Immunofluorescent microscopy demonstrated that both the MTMR6 CC and PH/G domains are required to co-localize MTMR6 to the plasma membrane with KCa3.1. These findings support a model in which two specific low affinity interactions are required to co-localize MTMR6 with KCa3.1: 1) between the CC domains on MTMR6 and KCa3.1 and (2) between the PH/G domain and a component of the plasma membrane. Our inability to detect significant interaction of the MTMR6 G/PH domain with phosphoinositides suggests that this domain may bind a protein. Identifying the specific binding partners of the CC and PH/G domains on other MTMs will provide important clues to the specific functions regulated by other MTMs as well as the mechanism(s) whereby loss of some MTMs lead to disease.
Choudhury
Papiya
P
Department of Medicine, The Skirball Institute, New York University School of Medicine, New York, NY 10016, USA.
Srivastava
Shekhar
S
Li
Zhai
Z
Ko
Kyung
K
Albaqumi
Mamdouh
M
Narayan
Kartik
K
Coetzee
William A
WA
Lemmon
Mark A
MA
Skolnik
Edward Y
EY
D000818:Animals; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D004285:Dogs; D015971:Gene Expression Regulation, Enzymologic; D051660:Intermediate-Conductance Calcium-Activated Potassium Channels / Q000737:chemistry; D018408:Patch-Clamp Techniques; D010716:Phosphatidylinositols / Q000737:chemistry; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D017027:Protein Tyrosine Phosphatases / Q000737:chemistry* / Q000378:metabolism; D054558:Protein Tyrosine Phosphatases, Non-Receptor; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D013379:Substrate Specificity
D051660:Intermediate-Conductance Calcium-Activated Potassium Channels; C496923:KCNN4 protein, human; D010716:Phosphatidylinositols; D011993:Recombinant Fusion Proteins; D010744:Phosphoric Monoester Hydrolases; D017027:Protein Tyrosine Phosphatases; D054558:Protein Tyrosine Phosphatases, Non-Receptor; C099588:myotubularin; C061816:phosphatidylinositol-3-phosphatase