Myotubularin-related protein 6 (MTMR6) is a catalytically active member of
the myotubularin (MTM) family, which is composed of 14 proteins. Catalytically
active myotubularins possess 3-phosphatase activity dephosphorylating
phosphatidylinositol-3-phoshate and phosphatidylinositol-3,5-bisphosphate, and
some members have been shown to form homomers or heteromeric complexes with
catalytically inactive myotubularins. We demonstrate that human MTMR6 forms a
heteromer with an enzymatically inactive member myotubularin-related protein 9
(MTMR9), both
in vitro and in cells. MTMR9 increased the binding of
MTMR6 to phospholipids without changing the lipid binding profile. MTMR9
increased the 3-phosphatase activity of MTMR6 up to 6-fold. We determined that
MTMR6 is activated up to 28-fold in the presence of phosphatidylserine
liposomes. Together, MTMR6 activity in the presence of MTMR9 and assayed in
phosphatidylserine liposomes increased 84-fold. Moreover, the formation of
this heteromer in cells resulted in increased protein levels of both MTMR6 and
MTMR9, probably due to the inhibition of degradation of both proteins.
Furthermore, co-expression of MTMR6 and MTMR9 decreased etoposide-induced
apoptosis, whereas decreasing both MTMR6 and MTMR9 by RNA interference led to
increased cell death in response to etoposide treatment when compared with
that seen with RNA interference of MTMR6 alone. Thus, MTMR9 greatly enhances
the functions of MTMR6.Myotubularin proteins are a family of 14 proteins with the canonical dual
specificity protein tyrosine phosphatase active site C
X5R
motif
(
1–
3).
Eight members of the myotubularin family possess catalytic activity,
dephosphorylating phosphatidylinositol 3-phosphate
(PtdIns-3-P)
4 and
phosphatidylinositol 3,5-bisphosphate (PtdIns-3,5-P
2) at the D-3
position, and six members are not catalytically active because they lack the
conserved cysteine residue in the protein tyrosine phosphatase motif that is
required for activity. Interest in this group of proteins originated from the
genetic evidence linking myotubularin, the founding member of this family, to
myotubular myopathy, an X-linked disorder characterized by severe hypotonia
and generalized muscle weakness
(
4). Subsequently, mutations in
MTMR2 and in its inactive binding partner MTMR13 were linked to a subset of
Charcot-Marie-Tooth disease type 4B, a demyelinating neurodegenerative
disorder (
5,
6).Despite near identical substrate specificity, biochemical and genetic
evidence supports the hypothesis that myotubularin proteins are not redundant
and have unique functions within cells
(
2,
7–
9).
The mechanisms by which loss of function of myotubularin proteins produce
diseases are not known. Current evidence supports the hypothesis that each
myotubularin protein regulates a specific pool of PtdIns-3-P and/or
PtdIns-3,5-P
2, which in turn regulates a variety of cellular
functions. Differences in tissue expression and subcellular localization play
a role in the specificity of different myotubularins
(
10–
15).The functions of myotubularin proteins are altered by the formation of
heteromers between catalytically active and inactive members of the family.
The initial biochemical purification of MTM1 demonstrated the presence of MTM1
homodimers and MTM1-3-phosphatase adapter protein (3PAP) heteromers
(
16), which was later
described as MTMR12 (
15,
17). MTMR2 was found to form
heteromers with MTMR5 (
13) and
MTMR13 (
18), and MTMR7 formed
heteromers with MTMR9 (
19). In
each case, a catalytically active myotubularin protein interacted with an
inactive protein. Heteromerization generated two important effects: increased
catalytic activity of the active component
(
13,
15,
19,
20) and targeting of the
heteromer to specific subcellular locations
(
15). Mutations in the
inactive member MTMR13 result in a similar phenotype in patients as the
mutations in its catalytically active binding partner MTMR2, indicating an
indispensable role for the catalytically inactive subunit
(
21).Myotubularin proteins can be grouped into subfamilies based on homology.
Closely related MTMR6, MTMR7, and MTMR8 comprise such a subfamily. We have
previously characterized the interaction between mouse MTMR7 and MTMR9
proteins (
19). In this report,
we characterize the interaction between human MTMR6 and MTMR9. MTMR6 and MTMR9
have been shown to form a heteromeric complex in mouse and
Caenorhabditis
elegans (
19,
22). MTMR6 has been shown to
inhibit the activity of a calcium-activated potassium channel (type KCa3.1)
(
23,
24). Two screening experiments
implicate MTMR6 as a regulator of apoptosis. By RNA microarray analysis,
increased MTMR6 expression was observed in B cell chronic lymphoid leukemia
cells with increased resistance to irradiation-induced apoptosis
(
25), whereas in an RNA
interference screen in HeLa cells, decreased MTMR6 expression promoted
apoptosis (
26).Here we show that MTMR6 interacts with MTMR9
in vitro and in human
cells. This interaction increases the phospholipid binding and enzymatic
activity of MTMR6
in vitro. Co-expression of either subunit in cells
dramatically increased the protein levels of the individual binding partners,
suggesting that heteromer formation increases the stability of the proteins.
Finally, MTMR9 was found to potentiate the effects of MTMR6 on apoptosis.
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