Co-expression of malin and laforin reduces glycogen accumulation
Overexpression of PTG leads to an increase in glycogen accumulation in tissue culture cells and ex vivo
organ models (41
). We sought to test the hypothesis that malin and laforin oppose the action of PTG. In order to gain insights into the mechanistic nature that malin and laforin play in glycogen metabolism, we measured glycogen accumulation in tissue culture cells expressing various combinations of PTG, laforin, and malin.
We utilized CHO-IR cells since they do not express PTG and produce a minimal amount of glycogen under normal tissue culture conditions () (37
). However, they exhibit a seven-fold increase in glycogen production when transfected with PTG () (37
). We transfected CHO-IR cells with a combination of PTG, laforin, and/or malin to determine how laforin and malin affect glycogen stores. Cells transfected with PTG and either laforin or malin yielded a 25% reduction in glycogen amounts compared to cells transfected with PTG and a vector control (). This reduction was not dependent on the phosphatase activity of laforin, because transfection with PTG and the catalytically inactive laforinC266S
yielded a similar reduction in glycogen stores (). However, the reduction was dependent on the protein-protein interaction domain of malin. Cells transfected with PTG and a LD disease mutation in the NHL domain of malin, malinE280K
, contained similar glycogen stores as vector control cells (). The effect of malin and laforin in concert was quite striking. Cells transfected with PTG, malin, and laforin contained a similar amount of glycogen as cells transfected with a vector control (). Therefore, malin and laforin each independently decreased PTG-stimulated glycogen accumulation, and cumulatively, they essentially eliminated PTG-stimulated glycogen accumulation.
Malin and laforin inhibit PTG/R5- and R6-stimulated glycogen accumulation
To determine if this result was a non-specific affect of overexpressing an E3 ubiquitin ligase, we transfected cells with PTG and MDM2, the E3 ligase that negatively regulates p53 (45
). While cells transfected with wild-type malin decreased PTG-stimulated glycogen stores, MDM2 did not reduce PTG-stimulated glycogen stores (). In a similar manner, we wished to ensure that simply overexpression of another gene or the addition of another plasmid did not reduce PTG-stimulated glycogen stores. Therefore, we transfected cells with PTG and either DJ-1, a Parkinson’s disease gene, or pEB6 (empty vector). In each case the transfected cells accumulated a similar amount of PTG-stimulated glycogen stores as PTG alone (). Therefore, the decrease in PTG-stimulated glycogen stores was not an artifact generated by a second vector, another gene being expressed, or expression of another E3 ubiquitin ligase.
PTG is one of five regulatory subunits that target PP1 to glycogen and modulate glycogen accumulation (36
). We investigated if malin and laforin specifically inhibit PTG-stimulated glycogen stores, or if they inhibit stimulated glycogen stores of other PTG family members. PTG and R6 (gene PPP1R3D
) are expressed in a wide range of human tissues, including the brain (36
). The other three PP1 targeting subunits display tissue specific expression patterns in skeletal muscle, and/or heart and liver tissue (48
). One such subunit is GL
), which is expressed in muscle and liver tissue (48
). To test our hypothesis, we transfected cells with PTG, R6, or GL
alone, and along with malin and laforin. As expected, PTG, R6, and GL
all stimulated increased glycogen accumulation (). Malin and laforin inhibited R6-stimulated glycogen accumulation, but they did not inhibit GL
-stimulated glycogen accumulation (). Therefore, malin and laforin inhibit PP1 regulatory subunit-stimulated glycogen accumulation of some PP1 regulatory subunits, PTG/R5 and R6, but not all of them. Interestingly these are the only two regulatory subunits that exhibit a wide expression pattern and are expressed in brain tissue (36
PTG is ubiquitinated and targeted for degradation in a laforin- and malin-dependent manner
We previously reported that malin promotes the ubiquitination and degradation of laforin and stated that this result, while correct, is in conflict with our understanding of LD genetics (26
). In addition, we postulated that malin likely promotes the degradation of another protein(s) and that this protein(s) may be a regulator of glycogen synthesis (26
To test if PTG could be a target of malin, we transfected CHO-IR cells with FLAG-tagged PTG along with myc-tagged malin and untagged laforin, immunoprecipitated PTG with α-FLAG beads, and probed for PTG expression with α-FLAG. We observed a slight reduction in PTG protein levels when cells were co-transfected with laforin alone compared to empty vector (), and consistently a greater reduction in PTG protein levels in cells co-transfected with malin alone (). When we transfected cells with FLAG-PTG and both malin-myc and laforin we observed a robust decrease in the protein levels of PTG (). To determine if the laforin-dependent reduction in PTG was dependent on the phosphatase activity of laforin, we transfected cells with FLAG-PTG, malin, and laforinC266S. Cells transfected with laforinC266S displayed similar decreased levels of PTG compared to cells transfected with wild type laforin (), demonstrating that this reduction was not dependent on the phosphatase activity of laforin. Therefore, malin and laforin in concert significantly reduced PTG protein levels, and this reduction is not dependent on the laforin’s phosphatase activity.
PTG protein levels are decreased in a malin-, laforin-, and proteasome-dependent manner
To determine if the interaction between malin and laforin was necessary to enhance the reduction in PTG, we transfected cells with FLAG-PTG and a mutant NHL-domain version of malin, malinE280K
-myc, which disrupts its interaction with laforin (26
). These cells accumulated a similar amount of FLAG-PTG as vector control cells and substantially more than cells transfected with wild-type malin (). In addition, cells transfected with FLAG-PTG, laforin, and the E3 ubiquitin ligases Pirh2 or MDM2 accumulated similar amounts of FLAG-PTG as cells transfected with no E3 ubiquitin ligase (). Thus, malin promotes a decrease in the accumulation of PTG, this decrease is dependent on the interaction between malin and laforin, and the decrease is not promoted by other E3 ubiquitin ligases.
Since malin is an E3 ubiquitin ligase (26
), it seemed plausible that malin could ubiquitinate PTG and target it for 26S proteasome-dependent degradation. To test if the decrease in PTG was dependent on the 26S proteasome, we performed similar experiments as above but in the presence and absence of the proteasome inhibitor MG132. Cells transfected with wild-type malin-myc and laforin and treated with MG132 accumulated similar PTG protein levels as vector control cells and substantially more than cells not treated with MG132 (, top panel). As a positive control, we monitored the protein levels of laforin in the presence and absence of MG132. As we would predict, since malin ubiquitinates laforin, laforin protein levels also increased in cells treated with MG132 (, bottom panel). Consistent with these results, when we preformed a denaturing immunoprecipitation of FLAG-PTG we found that cells co-transfected with malin-myc and laforin and treated with MG132 accumulated a significant amount of ubiquitin attached to PTG (). In addition, there was a substantial amount of FLAG-PTG that ran as a high molecular weight smear in the presence of MG132, similar to the detected ubiquitin smear (). However, treatment of cells with MG132 in the absence of transfected malin-myc also resulted in a high molecular weight smear of PTG and ubiquitin (). Similarly, we observed that transfection with laforin alone slightly decreased the protein levels of PTG (). This high molecular weight smear and the decrease in PTG in the absence of malin-myc is likely due to endogenous malin, since we detected malin mRNA in CHO-IR cells (data not shown). Cumulatively, these results strongly suggest that PTG is modified by the attachment of multiple ubiquitins in a malin-dependent manner, leading to the high molecular weight smear, and proteasome-dependent degradation.
To determine if PTG serves as a substrate for malin in vitro
, we performed in vitro
ubiquitination assays using recombinant, purified GST-malin-His6
S-labeled in vitro
translated PTG. Although we attempted this reaction several times under various conditions, including the conditions used for malin ubiquitination of 35
), we did not observe ubiquitination of 35
S-PTG (data not shown).
Given the lack of in vitro ubiquitination of PTG, we tested if malin and PTG directly interacted using recombinant, purified GST-malin-His6 and 35S-labeled in vitro translated PTG. Despite utilizing multiple conditions, we did not observe a direct interaction between malin and PTG (data not shown). In absence of evidence of a direct interaction, we asked if malin and PTG co-immunoprecipitate. We transfected cells with FLAG-malin and PTG-V5, treated cells with DMSO or DMSO + MG132, immunoprecipitated with α-V5, and blotted with α-V5 and α-FLAG. While malin did not coimmunoprecipitate with PTG under standard tissue culture conditions, malin did consistently coimmunoprecipitate with PTG in the presence of MG132 (). Therefore, while malin and PTG do not directly interact, they are in a complex when the 26S proteasome is inhibited.
Interestingly, PTG directly interacts with both laforin and malin via in vitro
binding experiments, and they interact in both yeast two-hybrid assays and overexpression studies in tissue culture cells (26
). While PTG interacts with laforin via
three exogenous assays, there have been no reports that endogenous PTG and laforin co-immunoprecipitate. Therefore, we tested if endogenous PTG and laforin interact. We immunoprecipitated PTG and blotted for PTG and laforin (). Endogenous PTG and laforin readily co-immunoprecipitated from tissue culture cells and this interaction was increased in the presence of the proteasome inhibitor MG132 (). Therefore, endogenous PTG and laforin are in a complex with each other, a complex likely involving a direct interaction between PTG and laforin. Since laforin and PTG are both degraded by the 26S proteasome, this interaction is increased when the proteasome is inhibited.
Given our glycogen assay results and the fact that PTG and laforin co-immunoprecipitate, it seemed plausible that laforin could act as a scaffold to allow malin to interact with and ubiquitinate PTG. Therefore, we performed the same in vitro
ubiquitin assay as above, but included recombinant laforin-His6
. Ubiquitination of 35
S-PTG was evaluated by Western blot analysis using avidin-HRP to detect biotinubiquitin and autoradiography to monitor the migration of 35
S-PTG. Malin ubiquitinated PTG in the presence of laforin, generating a high molecular weight shift in 35
S-PTG that co-migrated with high molecular weight ubiquitin (). If laforin was removed from the reaction, then PTG was not ubiquitinated (). Similarly, when malinE280K
, which does not efficiently bind laforin (26
), was utilized PTG was not ubiquitinated (). This result demonstrates that laforin acts as a scaffold between malin and PTG to allow malin to ubiquitinate PTG.
Cumulatively, these results demonstrate that malin ubiquitinates PTG and targets it for proteasome-dependent degradation, and this degradation decreases glycogen accumulation. In addition to PTG, malin ubiquitinates laforin and targets it for degradation (26
). Malin was also recently shown to ubiquitinate glycogen debranching enzyme (AGL/GDE) and target it for proteasome-dependent degradation (29
). The ubiquitination of AGL also occurred in the absence of a detectable interaction between malin and AGL. Similarly, we now demonstrate that malin ubiquitinates PTG in the absence of a detectable direct interaction. Therefore, we propose that laforin acts as a scaffold to tether malin in the vicinity of at least two substrates (and potentially others), AGL and PTG. In the absence of malin or laforin, PTG levels are increased and increased PTG levels cause glycogen synthase to be hyperactive (39
). Hyperactive glycogen synthase is one component driving the formation of LBs in LD patients. In support of this explanation, overexpression of glycogen synthase in muscle has been shown to produce a LB-like accumulation (51
), likely due to an imbalance between glycogen synthase and glycogen branching enzyme. These results suggest a new model when considering Lafora disease ().
Model of the mechanisms of Lafora disease
As illustrated in , we propose that phosphate is incorporated in glycogen, as a by-product of normal glycogen synthesis. There are reports of phosphate being present in mammalian glycogen (52
), and phosphorylation of plant starch is a highly coordinated mechanism (53
). In addition, LBs contain excess phosphate compared to glycogen (10
). Once a glycogen particle forms, PTG and AGL are both targeted to the particles via
their CBMs, and are both involved in glycogen synthesis (37
). Laforin too is targeted to glycogen via
its CBM and once attached it dephosphorylates glycogen, an activity novel to laforin and laforin-like phosphatases (19
). After laforin has bound glycogen, malin directly interacts with laforin and ubiquitinates laforin, AGL, and PTG (26
). This ubiquitination releases all three proteins from glycogen and both targets them for proteasome-dependent degradation and allows glycogen metabolism to proceed normally. Thus, laforin plays a role in regulating multiple proteins that drive glycogen metabolism. In support of this role for laforin, data from multiple mouse models demonstrate that laforin protein levels closely correlate with the levels of intracellular glycogen (57
). While our model does not account for all aspects of glycogen metabolism, it provides a foundation in understanding multiple facets of Lafora disease.
In this model, malin in essence behaves as a uni-directional signal, and laforin acts as both an activator and repressor of proper glycogen metabolism. The CBM of laforin localizes it to glycogen and positions laforin to dephosphorylate glycogen; this dephosphorylation is an “activator” of glycogen metabolism. In the absence of this dephosphorylation, some aspect of glycogen synthesis is “impaired”, possibly branching, and this is one component that leads to a LB. After dephosphorylation, laforin and additional proteins (such as PTG and AGL) must be degraded or they inhibit proper glycogen metabolism. Thus, laforin is analogous to previously described “activation by destruction” transcriptional activators (58
), where laforin provides a positive “signal” for glycogen metabolism but then must be degraded for glycogen metabolism to continue. If laforin is lacking then glycogen becomes hyperphosphorylated, glycogen debranching enzyme (AGL/GDE) is overactive which results in fewer branches, and glycogen synthase is overactive due to increased phosphatase activity of PP1-PTG and that also leads to fewer branches. These two characteristics, increased phosphorylation and decreased branches, are both hallmarks of LBs.
This model ascribes laforin two functions in suppressing LD, 1) dephosphorylation of glycogen in order to inhibit LB accumulation (19
), and 2) it recruits malin to the site of glycogen accumulation so that malin can ubiquitinate PTG, AGL, and laforin (26
). These concerted events target PTG, AGL, and laforin for proteasome-dependent degradation, thereby inhibiting LB accumulation and allowing glycogen metabolism to proceed normally. The two roles for laforin and one for malin in inhibiting LD are consistent with patient data which demonstrate that mutations in laforin results in a more severe phenotype and shorter patient life-span than mutations in malin (61
). Thus, the more biochemical interactions and mechanisms we uncover concerning malin and laforin, the more we understand this complex, neurodegenerative disease.