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Originally published In Press as doi:10.1074/jbc.M606344200 on August 16, 2006

J. Biol. Chem., Vol. 281, Issue 42, 31762-31769, October 20, 2006
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Specificity of the Myotubularin Family of Phosphatidylinositol-3-phosphatase Is Determined by the PH/GRAM Domain*

Papiya Choudhury{ddagger}§1, Shekhar Srivastava{ddagger}§1, Zhai Li{ddagger}§, Kyung Ko{ddagger}§, Mamdouh Albaqumi§, Kartik Narayan||, William A. Coetzee{ddagger}**, Mark A. Lemmon||, and Edward Y. Skolnik{ddagger}§2

From the Departments of {ddagger}Medicine and **Pediatric Cardiology, the Division of Nephrology, and §The Skirball Institute, New York University School of Medicine, New York, New York 10016 and the ||Department of Biochemistry and Biophysics, University of Pennsylvania Medical Center, Philadelphia, Pennsylvania 19104

Received for publication, July 3, 2006 , and in revised form, August 1, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Myotubularins are a large family of lipid phosphatases that specifically dephosphorylate the D3 position of PI(3)P3 and PI(3,5)P2 (1-3). Fourteen MTMs have been identified in mammals, and although eight have been shown to have catalytic activity, six MTMs encode for proteins that contain a mutation in the catalytic active site and lack phosphatase activity. However, although most phosphatase active MTMs have nearly identical substrate specificity, both genetic and biochemical studies have indicated that many MTMs perform unique functions and are not functionally redundant with other MTMs (2, 4, 5). MTM1, the founding member of this family, was identified genetically as the gene that is mutated in X-linked congenital myotubular myopathy (6). Subsequently, MTMR2 and MTMR13 were found to be mutated in a subset of patients with Charcot-Marie-Tooth disease 4B (7, 8). In addition, MTMR6 and MTMR9 were identified genetically to be required in Caenorhabditis elegans for endocytosis (9, 10).

Although a great deal has been learned about MTMs over the past several years, for the most part the specific biological processes regulated by MTMs are not known. In addition, nothing is known about the perturbations that arise when MTMs are mutated in diseases such as X-linked myotubular myopathy and Charcot-Marie-Tooth disease 4B, and little is still known about the mechanism of MTM regulation or what accounts for the specific function of the different MTMs. Recent evidence has indicated that some of the specific functions of MTMs may be related to their unique cell expression patterns and more importantly to their localization to unique subcellular compartments within a given cell (10-14). In this scenario, each MTM would function in the regulation of only specific pools of PI(3)P in specific subcellular contexts.

An important insight into the regulation of MTMs as well as the mechanism for targeting MTMs to specific subcellular compartments was the observation that members of the phosphatase active MTMs specifically heterodimerize with phosphatase inactive members via coiled-coil (CC) domains in the carboxyl terminus of each protein (11, 12, 14-16). Although one important function for heterodimerization is to increase the phosphatase activity of the phosphatase live MTM in the complex, the phosphatase inactive MTM also functions as an adaptor and contributes to the targeting of a phosphatase live MTM to specific subcellular localizations in the cell. For example, the interaction of MTMR2 (active) with MTMR5 (inactive) and the interaction of MTM1 (active) with MTMR12 (inactive) play an important role in the subcellular localization of the phosphatase active MTM in the complex (12, 14).

In addition to containing CC domains, all MTMs contain a PH/GRAM (G) domain. Although this domain was originally identified on the basis of homology from bioinformatics between three proteins (glucosyltransferases, Rab-like GTPase activators, and myotubularins) (17), the crystal structure of MTMR2 revealed for the first time that the MTM GRAM domain is part of a larger motif that encompasses a PH domain, which led to the subsequent renaming of this domain in MTMs as a PH/G domain (18, 19). Subsequently, several studies have demonstrated that the PH/G domain in a number of different MTMs bind phosphoinositides and also function to localize MTM to different subcellular compartments in the cell (11, 13, 20).

We recently found that the calcium-activated potassium channel KCa3.1 requires PI(3)P for activity and is specifically inhibited by MTMR6 (21-23). These studies demonstrated that interaction between the CC domain of MTMR6 with the CC domain in the carboxyl terminus of KCa3.1 is critical for the inhibition of KCa3.1 and proposed that interaction between the CC domains on KCa3.1 and MTMR6 functions to localize MTMR6 at the PM where it then dephosphorylate PI(3)P leading to KCa3.1 inhibition. We have now extended these findings to demonstrate that the MTMR6 PH/G domain is also critical for inhibition of KCa3.1 by functioning together with the CC domain to localize MTMR6 with KCa3.1 in the plasma membrane (PM). Because KCa3.1 is the first and only bona fide MTM target, these studies provide an important insight into the general function of these domains across MTM family members.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Constructs—CHO-KCa3.1 cells, a stable Chinese hamster cell line that overexpress FLAG-tagged KCa3.1, have been described previously (21). Chimeric MTMs were generated in which the MTMR6 G/PH or CC (aa 1-106 or 512-621) domains were replaced with the corresponding domains on MTMR2, -R3, or -R8 by overlapping PCR and cloned in into the vector pEGFP (Clontech). See the schematic in Fig. 1 for a summary of the constructs and the exact amino acid substitutions.

Cell Culture, Transfection, and Patch Clamping—GFP-tagged constructs were transfected into CHO-KCa3.1 cells using FuGENE (Roche Applied Science), and whole cell patch clamping was performed 48 h after transfection on GFP-positive cells as described (21). Patch clamp pipettes had resistances ranging between 2.2 and 3.5 megohms. Current-voltage (I-V) relationship was measured using ramp voltage clamp protocols (at 15-s intervals) from a holding potential of -70 mV to -120 mV followed by ramp depolarization to +60 mV (symmetrical ramp rate of 0.18 mV·ms-1). The current-voltage relationship was obtained by plotting the current during the depolarizing ramp phase as a function of the corresponding voltage. Membrane currents were filtered (-3 dB at 1 kHz) and digitized at 10 kHz (pClamp 9.2 with Digidata 1200 ADC interface, Axon Instruments). Cell capacitance and pipette series resistances were compensated (usually >80%), and these were obtained using the "membrane test" function of Clampex. Whole cell current density was expressed as pA/pF (picofarad).


Figure 1
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  		FIGURE 1.
Schematic of the chimeric MTMs. Amino acids that were substituted are as follows: MTMR2/6CC (1-504 aa of MTM2 and 507-621 aa of MTMR6); MTMR2/6PH/G (1-67 aa of MTMR2 + 10-106 aa of MTMR6 + 192-643 aa of MTMR2); MTMR2/6PH/G&CC (1-67aa of MTMR2 + 10-106 aa of MTMR6 + 192-507 aa of MTMR2 + 506-621 aa of MTMR6); MTM1/6CC (1-540 aa of MTM1 and 506-621 aa of MTMR6); MTMR8/6CC (1-502 aa of MTMR8 and 506-621 aa of MTMR6); MTMR8/6PH/G (1-106 aa of MTMR6 and 107-704 aa of MTMR8); MTMR8/6PH/G&CC (1-106 aa of MTMR6 + 107-502 aa of MTMR8 + 506-621 aa of MTMR6); MTMR8/1PH/G&6CC (1-11 aa of MTMR8 + 24-162 aa of MTM1 + 126-502 aa of MTMR8 + 506-621 aa of MTMR6). PT, phosphatase domain.

 
Immunofluorescence—To determine whether any of the MTMs co-localized with KCa3.1 at the plasma membrane, MDCK cells were co-transfected with GFP-tagged MTMs together with hemagglutinin epitope-tagged KCa3.1 (HA-KCa3.1) in which the HA epitope was inserted into the extracellular loops between S3 and S4. Previous studies have demonstrated that KCa3.1 containing this HA epitope functions normally and can be specifically detected at the PM by performing immunofluorescence on nonpermabilized cells (22, 24). 48 h after transfection, cells were fixed with 4% paraformaldehyde, and KCa3.1 was detected in nonpermeabilized by staining with antibodies to HA followed by Cy3-labeled anti-mouse IgG. Cells were then visualized for GFP and Cy3 using an inverted laser-scanning confocal microscope (Leica SP2 Microsystems) as described previously (21). TIFF images were processed for both GFP-MTMs and Cy3-KCa3.1 with Adobe Photoshop 6.0, and merged images were generated to assess co-localization.

Binding Studies of the PH Domain of MTMR6 to Phosphoinositides—The GST-PH domain of MTMR6 (aa 1-106) was expressed in Escherichia coli and purified as described previously. Lipid overlay assays were performed on PIP Strips purchased from Echelon Biosciences. To determine whether GST-MTMR6(PH) binds any PIs, PIP Strips were incubated with 10 µg of GST-MTMR6(PH). After washing, bound GST-MTMR6 was determined by staining with anti-GST antibodies according to manufacturer's protocols.

Surface Plasmon Resonance (SPR) of Phosphoinositide Binding—Affinity studies by SPR were performed using a Biacore instrument as described previously (25). Briefly, phosphatidylcholine vesicles with or without 3% (mol/mol) added PI were immobilized on an L1 sensor chip, and binding of various concentrations of GST-MTMR6(PH) was assessed to phosphatidylcholine alone (background) or to dioleoylphosphatidylcholine containing a PI.

Western Blot—SDS-PAGE and Western blotting were performed as described previously (26). GFP-tagged MTMs were visualized using an anti-GFP antibody purchased from Clontech.


Figure 2
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  		FIGURE 2.
MTMR6, but not other MTMs, inhibits KCa3. 1 channels. CHO cells overexpressing KCa3.1 (CHO-KCa3.1) were transfected with GFP control (A) or GFP-MTM constructs (B-F) as indicated, and whole cell patch clamping was performed 48 h after transfection using green fluorescent cells. Shown are the current-voltage plots of KCa3.1 cells before and after treatment with Tram-34 (at 1 µM for 3-5 min), demonstrating the identity of the current as being formed by KCa3.1 channels. The K+ current displays a reversal potential of -73 mV (not corrected for the liquid junction potential, calculated to be about 13 mV), which is similar to the calculated reversal potential of -84 mV at an extracellular K+ of 5 mM. G, bar graph summary of experiments A-F of Tram-34 inhibitable current plotted at -120 and +60 mV; n = 8 cells for each group. Only MTMR6 inhibits KCa3.1.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
MTMR6 Is the Only Phosphatase Active MTM That Inhibits KCa3.1—All MTM family members contain a similar overall structure and are composed of PH/G, phosphatase, and CC domains (1, 4). To determine whether other MTMs inhibit KCa3.1, GFP-tagged MTMs were transfected into CHO cells that overexpress KCa3.1 (CHO-KCa3.1), and cells positively identified as being transfected by green fluorescence were subjected to whole cell patch clamp experiments as described previously (21, 22). We found that of the MTMs tested, only MTMR6 inhibited KCa3.1 (Fig. 2). There are eight mammalian MTMs that contain active phosphatases, and these can be further subdivided into three major subfamilies consisting of: MTMR6, -R7, and -R8; MTMR3 and -R4; and MTM1, -R2, and -R3 (2, 27). Although we have previously found that MTM1 does not inhibit KCa3.1, we now show that representative members of MTMs from all the MTM subfamilies including MTMR2, MTMR3, and MTMR8 also do not inhibit KCa3.1 (Fig. 2). These findings indicate that of the MTMs tested, KCa3.1 is specifically inhibited only by MTMR6.

Replacement of the MTMR2 and MTMR8 CC Domains with the CC Domain of MTMR6 Did Not Enable These Chimeric MTMs to Inhibit KCa3.1—We showed previously that although MTM1 does not inhibit KCa3.1, substitution of the MTMR1 CC domain with the CC domain of MTMR6 creates a chimeric MTM that inhibits KCa3.1 (21). Based on this finding, we proposed that the CC domain of MTMR6 would be sufficient to dictate MTM specificity for a particular target (KCa3.1) and that CC domains on the different MTMs would be critical in determining the specific target regulated by a given MTM. For example, if binding of only the MTMR6 CC domain to the CC domain on KCa3.1 was sufficient to recruit MTMR6 to KCa3.1, only MTMR6 would be recruited to KCa3.1 where it could then dephosphorylate a specific pool of PI(3)P leading to KCa3.1 inhibition. In an attempt to generalize these findings to other MTMs, we created MTMR2 and MTMR8 chimeras in which the CC domains of these MTMs were substituted for the CC domain of MTMR6. Surprisingly, neither the MTMR2/MTMR6CC or MTMR8/MTMR6CC chimeras inhibited KCa3.1 channel activity, despite the fact that these constructs were expressed equally as well as the MTM1/MTMR6CC chimera and contained the replacement of the same amino acids from MTMR6 (Fig. 3, A and D). Thus, these findings indicate that the CC domain alone is not sufficient to dictate the specific regulation of KCa3.1 by an MTM.


Figure 3
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  		FIGURE 3.
Both the CC and PH/G domains of MTMR6 mediate the specific inhibition of KCa3.1 by MTMR6. A-C, CHO-KCa3.1 were transfected with GFP-MTM constructs as indicated, and whole cell patch clamping was performed as described in the legend for Fig. 2. Shown are the bar graph summaries of Tram-34 inhibitable current plotted at -120 and +60 mV, n = 8 cells. D, anti-GFP Western blots of lysates from cells transfected in A-C demonstrating that differences in expression of the various constructs does not account for the results obtained. IB, immunoblot.

 
The MTMR6 G/PH Domain Together with Its CC Domain Plays a Critical Role in the Inhibition of KCa3.1—To assess whether the PH/G domain is also important for KCa3.1 inhibition by MTMR6, we generated an MTMR6 with a point mutation in the PH/G domain at position 27 (28). This mutation was initially identified in patients with inactivating mutations in MTM1 and has been shown to inhibit binding of the MTM1 PH/G domain to phosphoinositides. We found that mutation of the analogous amino acid in the PH/G domain of MTMR6 (MTMR6(G27E)) interfered with the inhibition of KCa3.1 by MTMR6 suggesting that the MTMR6 PH/G domain is also essential for KCa3.1 inhibition by MTMR6 (Fig. 3B).

One of the difficulties in interpreting studies that rely on point mutations in the PH/G domain is that some of these mutations may also affect MTM phosphatase activity or protein folding (13, 28). Therefore, there is no reliable way to distinguish whether a point mutation uncouples a specific function of the PH/G domain or whether creation of a mutation interferes with a more general MTM function such as phosphatase activity; MTMR6 phosphatase activity is required for MTMR6 to inhibit KCa3.1 (21). Thus, the MTMR6 PH/G domain function was also assessed in the context of chimeric MTMs. We reasoned that the role of the MTMR6 PH/G domain could be definitively addressed if replacement of the MTMR2 or MTMR8 PH/G domain with that of MTMR6 resulted in a gain of function that now resulted in inhibition of KCa3.1. These studies demonstrated that chimeric MTMR2 and -R8 proteins containing the MTMR G/PH and CC domains inhibited KCa3.1 to a similar degree as did MTMR6 (Fig. 3C), whereas substitution of either domain alone did not (Fig. 3, A and B). The inability of an MTM to inhibit was not related to protein expression, as MTMs that failed to inhibit expressed equally well as MTMs that did inhibit (Fig. 3D). These findings indicate that although the phosphatase domain is redundant among the various MTMs tested, both the MTMR6 PH/G and CC domains perform unique functions for MTMR6 to inhibit KCa3.1.

The MTMR6 PH/G and CC Domains Are Required to Colocalize MTMR6 at the Plasma Membrane—We next tested the possibility that the two low affinity interactions are required to localize MTMR6 at the plasma membrane associated with KCa3.1 where it could then dephosphorylate a specific pool of PI(3)P leading to the inhibition of KCa3.1. GFP-tagged MTMs were co-transfected with FLAG-tagged KCa3.1, and co-localization was assessed by immunofluorescence using confocal microscopy. Although the majority of overexpressed GFP-MTMs were cytosolically localized, possibly as a result of overexpression, a portion of the overexpressed MTMs or chimeric MTMs that inhibited KCa3.1 also co-localized with KCa3.1 at the plasma membrane (Fig. 4). For example, although wild type MTMR6 co-localized with KCa3.1 at the PM (Fig. 4A), neither MTM1, MTMR2, MTMR3, nor MTMR8 co-localized with KCa3.1 (Fig. 4C, panels a, c, g, and k). However, for MTMR2 and MTMR8, replacement of both the CC and PH/G domains with the analogous domains on MTMR6 now resulted co-localization of a portion of the chimeric MTM with KCa3.1 (Fig. 4C, panels f and j), whereas substitution of either domain alone did not lead to co-localization with KCa3.1 (Fig. 4C, panels d, e, h, and i). In addition, a chimeric MTM1 containing only the MTMR6 CC domain, which we found previously was sufficient to inhibit KCa3.1, also co-localized with KCa3.1 (Fig. 4C, panel b). This finding suggests that the function of the MTM1 G/PH domain, unlike the G/PH domains of MTMR2 and MTMR8, is redundant with the PH/G domain of MTMR6. Consistent with this latter idea, we found that a chimeric MTMR8 containing the MTM1 G/PH domain together with the MTMR6 CC domain also inhibited KCa3.1 (Fig. 3C). The importance of the MTMR6 G/PH domain to the localization of MTMR6 to the plasma was further substantiated by the finding that MTMR6(G27E) failed to co-localize with KCa3.1 at the plasma membrane (Fig. 4B).


Figure 4
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  		FIGURE 4.
Both the CC and PH/G domains of MTMR6 are required to specifically co-localize MTMR6 with KCa3.1 at the plasma membrane. MDCK cells were transfected with exofacially tagged HA-KCa3.1 together with GFP-wild type or chimeric MTMs as indicated. Nonpermeabilized fixed cells were stained with anti-HA antibodies and visualized with Cy3-labeled anti-mouse IgG. Confocal merged images of photographs taken on the green and red channels are shown. Co-localization is visualized as yellow. Note that there is a perfect correlation between the MTM constructs that inhibit KCa3.1 in Fig. 2 and their co-localization with KCa3.1. A, MDCK cells co-transfected with HA-tagged KCa3.1 and wild type MTMR6. Shown are GFP-MTMR6 (MTMR6 (green)), exofacially tagged KCa3.1 (KCa3.1 (red)), and the merged image (MTMR6 + KCa3.1 (yellow)). B, merged images of MDCK cells transfected with MTMR6 containing an inactivating mutation in the PH/G domain (MTMR6(G27E)) or deleted of its CC domain (MTMR6-CC). Note that neither mutant co-localizes with KCa3.1. C, experiments, similar to those in A, of other MTMs that fail to inhibit KCa3.1. Merged images of MDCK cells transfected with HA-tagged KCa3.1 and MTM1 (a), MTMR2 (c), MTMR8 (g), or MTMR3 (k). These are the same MTMs in which the MTMR6 CC and/or PH/G domains were substituted for similar domains. +MTMR6 CC, chimeric MTM containing the MTMR6 CC domain (b, d, h); +MTMR6 PH/G, chimeric MTM containing the MTMR6 PH/G domain (e, i); +MTMR6 CC, + MTMR6 PH/G & CC, chimeric MTM containing the MTMR6 PH/G and CC domains (f, j).

 
The MTMR6 PH/G Domain Binds PIs with Low Affinity—The requirement for the MTMR6 PH/G domain for PM localization suggests that the PH/G domain interacts with a component of the PM. Previous studies have indicated that various MTM PH/G domains bind several different PIs with some specificity, although the affinity of interaction has not been rigorously assessed (11, 13, 20). Probing dot blots with a GST·MTMR6·PH/G fusion protein, we found that the PH/G domain of MTMR6 bound promiscuously to a number of different PIs including PI(4)P, PI(5)P, PI(3,4)P2, PI(3,5)P2, PI(4,5)P2, PI(3,4,5)P3, and phosphatidic acid (Fig. 5).

Although probing dot blots is useful in assessing which PIs a given PH/G domain may bind, this process may detect very low affinity interactions that are not biologically relevant. Thus, to directly assess the affinity of interaction, lipid vesicles were immobilized on a commercial biosensor chip surface, and the phenomenon of SPR was used to detect the binding of proteins to this surface when GST-MTMR6(G/PH) flowed over the surface by Biacore. The output from the SPR apparatus, measured in resonance units, is directly proportional to the mass of protein bound to the experimental binding surface. Specific binding of the MTMR6 G/PH domain to a PI was determined by measuring the difference, in resonance units, between MTMR6-binding vesicles containing phosphatidylcholine alone or those containing phosphatidylcholine together with the PI as indicated. Binding of PIs to the MTMR6 G/PH domain was not detected to any appreciable degree (data not shown). Based on previous studies,4 these findings indicate that the affinity of interaction between the MTMR6 and the PIs shown on the dot blot in Fig. 5 is less than 1 mM.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Although it is widely appreciated that MTMs regulate specific biological processes, the mechanism for this specificity has not been clear. Our finding that KCa3.1 channel activity is inhibited by MTMR6, but not by other MTMs, provided us with a unique opportunity to determine the mechanism whereby an MTM specifically regulates a target. These studies demonstrate for the first time that the G/PH domain is critical for mediating the specific regulation of a target by an MTM. MTMR6 inhibition of KCa3.1 is dependent upon both its CC and PH/G domains, which function together to localize MTMR6 with KCa3.1 at the PM where it can then dephosphorylate a specific pool of PI(3)P leading to the inhibition of KCa3.1. Moreover, the finding that the phosphatase domain is functionally redundant between different MTMs is consistent with in vitro data demonstrating that all phosphatase active MTMs dephosphorylate the D3 position of PI(3)P or PI(3,5)P2, which suggests that targeting of an MTM to a specific subcellular membrane leading to the dephosphorylation of a specific subcellular pool of PI(3)P determines the unique functions of the different MTMs (only MTMR6 co-localizes with KCa3.1 at the PM).

Our studies reported here demonstrate that the MTMR6 PH/G domain must function in concert with its CC domain to target MTMR6 to KCa3.1 at the plasma membrane. This conclusion is based on several findings including: 1) an inactivating point mutation in the MTMR6 G/PH domain does not inhibit KCa3.1 and fails to localize at the PM with KCa3.1; 2) chimeric MTMR2 and MTMR8 proteins containing only an MTMR6 CC or G/PH domain do not inhibit or co-localize with KCa3.1 at the PM; whereas 3) chimeric MTMR2 and MTMR8 proteins containing both the MTMR6 CC and PH/G domains both inhibit KCa3.1 and co-localize with KCa3.1 at the PM.

Because PH domains are best known for their function of binding PIs and localizing proteins to membranes, the resemblance of the GRAM domain to PH domains reinforced the idea that the G/PH domain in MTMs also functions to localize MTMs in membranes. Our findings reported here are consistent with this idea. However, the inability of either the MTMR2 or MTMR8 PH/G domain to substitute for that of MTMR6 indicates that the PH/G domains on these and other MTMs likely target a particular intracellular membrane that is different from that of MTMR6. In the case of the MTMR6 PH/G domain, our findings would suggest that MTMR6 PH/G domain functions specifically to bind a component in the PM that other MTM PH/G domains, including MTMR2, -R3, and -R8, are unable to bind. In contrast, the finding that the MTM1 PH/G domain is functionally redundant with that of MTMR6 with regard to inhibiting KCa3.1 and mediating recruitment to the PM would suggest that both the MTM1 and the MTMR6 PH/G domains bind a similar component in the PM.


Figure 5
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  		FIGURE 5.
MTMR6 PH/G domain binds promiscuously to a number of different phosphoinositides. Binding of the MTMR6 PH/G domain to various phosphorylated PIs was assessed by lipid overlay assays. GST-MTMR6(PH/G) was incubated with PIP Strips purchased from Echelon Biosciences. After washing, bound MTMR6(PH/G) bound to PIs was assessed using anti-GST antibodies and visualized using horseradish peroxidase-conjugated anti-rabbit IgG.

 
A major unresolved question is what the MTMR6 PH/G domain is binding in the PM. Consistent with the known phosphoinositide binding function of more classic PH domains, a number of studies have now demonstrated binding of MTM PH/G domains to PIs (11, 13, 20). In particular, MTM1, MTMR2, and MTMR3 have all been shown, by lipid dot blots (protein lipid overlay assays), to bind promiscuously to a number of PIs (11, 13, 20). However, although lipid dot blots are useful in screening for which of the PIs may bind a particular PH domain, they may also identify many low affinity interactions that are not biologically meaningful. Thus, additional studies have tried to confirm the biological relevance of these interactions using more specific binding and functional assays. These studies demonstrate that the PH/G domain of MTM1 preferentially binds PI(3,5)P2, whereas the PH/G domain of MTMR3 preferentially binds PI(5)P (13, 20). In addition, the PH/G domain of MTMR2 has been shown to mediate the redistribution of MTMR2 to PI(3,5)P2-containing vesicles in osmotically shocked cells (11). We found by lipid dot blots that the PH/G domain of MTMR6 also bound promiscuously to a number of different phosphoinositides. However, we did not detect binding of the MTMR6 PH/G domain to any PI, using Biacore, which indicates that the affinity for interaction between the MTMR6 PH/G domain and PIs is of very low affinity and is likely less then 1 mM (25, 29). Thus, this finding indicates that if binding of the MTMR6 PH/G domain to a PI in the plasma membrane is critical for PM targeting, it must cooperate with other low affinity interactions, which could explain why interaction between the CC domain of MTMR6 and the CC domain of KCa3.1 is also required for MTMR6 recruitment to the PM.

It is also possible, however, that the PH/G domain of MTMR6 binds another component in the PM, such as a protein. Unlike the case of some PH domains that bind PIs with high affinity (such as AKT and PLC{delta}, which bind, respectively, to PI(3,4,5)P3 and PI(4,5)P2 and are sufficient to target localization to a membrane compartment (29, 30)), it has recently become apparent that most conventional PH domains either do not bind PIs or bind only with low affinity (25, 29). Nevertheless, even PH domains that bind PIs with low affinity may play a critical role in membrane targeting, although correct subcellular targeting requires that the PH domain bind a second ligand. For example, in the case of Golgi targeting by the OSBP (oxysterol-binding protein) and FAPP1 (PI-four-phosphate adaptor protein 1) PH domains, low affinity binding to both an activated G protein, such as ARF (low affinity), and phosphoinositides (PI(4)P) is required (31, 32). Although lipids contribute to binding, interaction with a specific protein localized to a specific subcellular compartment (ARF) defines the subcellular localization of these PH domains. In addition, the finding that the PH domains of Osh1p and Skm1p have identical phosphoinositide binding specificities, yet localize to distinct subcellular compartments, provides further support for this model; Osh1p localizes to the Golgi, whereas Skm1p localizes to the PM. We would like to propose that the PH/G domain in MTMs may function like the PH domains of Skm1p and Osh1P. For example, the PH/G domain of MTMR6 may function like Skm1p to localize to the PM by binding a PM protein in the context of low affinity interaction with a PI, whereas the PH/G domain of another MTM may function like Osh1p to localize to the Golgi by binding a Golgi specific proteins. This model would also be consistent with experimental evidence suggesting that MTMs bind PIs with only low affinity; the crystal structure of the MTMR2 PH/G domain did not contain PIs even when exposed to very high concentrations of PI (1, 18). Moreover, G/PH domains lack the positive charges in the b1-b2 loop, which have been shown to be critical for high affinity binding to PIs (29, 33). The challenge in the future will be to definitively address whether low affinity interactions between MTM PH/G domains and proteins localized to specific subcellular compartments play an important role in the subcellular targeting of MTMs. Finding creative means to identify these interactions will likely be important, because the affinity of interaction will most likely not be sufficient to identify these interactions using standard approaches.


   FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Co-first authors. Back

2 To whom correspondence should be addressed: The Skirball Inst., New York University School of Medicine, 540 First Ave., New York, NY 10016. Tel.: 212-263-7458; Fax: 212-263-8951; E-mail: Skolnik{at}saturn.med.nyu.edu.

3 The abbreviations used are: PI, phosphatidylinositol; MTM, myotubularin; PH/G, pleckstrin homology/GRAM (glucosyltransferases, Rab-like GTPase activators, and myotubularins); GST, glutathione S-transferase; aa, amino acid(s); HA, hemagglutinin; GFP, green fluorescent protein; CC, coiled-coil; PM, plasma membrane; CHO, Chinese hamster ovary; SPR, surface plasmon resonance; MDCK, Madin-Darby canine kidney cells. Back

4 M. A. Lemmon, unpublished observation. Back


   ACKNOWLEDGMENTS
 
We thank Steve Hubbard (NYU School of Medicine) for helpful discussions, Dr. K. George Chandy (University of California, Irvine) and Heike Wulff (University of California, Davis) for Tram-34 and Dr. Francis Barr (Max Planck Institute of Biochemistry, Martinsried, Germany) for the GST-MTMR6 construct.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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