Hello, I copy and paste the article to save image space. Can you please summarize what this paper is about and elaborate the result? The discussion should explain what this article is about. Please summarize the paper and its conclusion as if your explaing step by step to the class.
Transcribed Image Text:movement of the three cytoplasmic domains, N (nucleotide binding), P (phosphorylation), and A
(actuator) (37). A recent study (28) suggests that the NH2 terminus may act as an auto-regulatory
domain, modulating E1/E2 conformational transition. In El conformation, the NH2 terminus is in
association with the first cytoplasmic loop of Na,K-ATPase. Transition from E1 to E2 conformation
may release the NH2 terminus from its interaction with the first cytoplasmic loop of Na,KATPase,
thus making the NH2 terminus available for interaction either with the InsP3R directly or with a
protein bridging between Na,K-ATPase and InsP3R. Such an effect could explain why truncation of
the first 32 amino acids of the NH2 terminus will prevent ouabain-induced Ca2 oscillations. An
alternative explanation is that NH2-terminal truncation has displaced the E1 3 E2 conformation of
Na,K-ATPase in favor of E1 (8, 38). The concept that Na,K-ATPase may, in addition to its function
as an ion pump, also act as a signal transducer, is now rapidly evolving (2-5). Most previous studies
on the signaling role of Na,K-ATPase have been carried out on cardiac myocytes (for a review, see
Ref. 39). In these cells, activation of Src kinase was found to be the primary event in the signaling
cascade initiated by the ouabain/Na,K-ATPase complex, and downstream effects included
phosphorylation of epidermal growth factor receptor and activation of the mitogen-activated protein
kinase pathway. Since cardiac myocytes exhibit spontaneous Ca2 sparks of high frequency, it has not
yet been possible to establish whether Na,K-ATPase/InsP3R-triggered Ca2 oscillations also occur in
these cells. Another model where Na,K-ATPase-mediated Ca2 signaling is dependent on a close
proximity between the plasma membrane and ER Ca2 stores has been presented previously (40). In
this model, Na,KATPase 2- and 3-subunits modulate Ca2 release from ER via local changes in
intracellular Na concentration. Our study was performed on cells expressing only the 1-subunit of
Na,KATPase, and the results from the protocols with the truncated NKA1.M32 imply that increased
intracellular Na concentration was, in our model, not the main cause of ouabain-induced Ca2
oscillations. Furthermore, we previously established that increasing intracellular Na, by lowering
extracellular K to the same extent as 250 M ouabain, is not sufficient to elicit a Ca2 oscillatory
response (2). Ca2 oscillations trigger such universal processes as fertilization, cell division, cell
differentiation, and apoptosis (41). The specificity of this signal is encoded by the frequency and/or
the amplitude of the Ca2 oscillations (42). In the case of G-proteincoupled receptors, Ca2 oscillations
generally have a periodicity of seconds (30, 43). The highly regular ouabain/Na,KATPase-induced
Ca2 oscillations displayed a longer periodicity in the minute range and should thereby provide a
mechanism to ensure a different kind of physiological readout. Endogenous ouabain levels are high
during pregnancy and in the perinatal period (44, 45). Thus, it is of great physiological interest that
the ouabain-induced slow Ca2 oscillations were found to activate NF-B. This pluripotent
transcription factor activates genes modulating cell proliferation, apoptosis, and development as well
as responses of the immune system (46). Cells expressing GFP-NKA1.M32 did not respond to
ouabain with Ca2 oscillations. In these cells, ouabain did not activate NF-B. These findings support
the physiological significance of a signaling microdomain containing Na,K-ATPase 1-subunit and
InsP3R as a trigger mechanism for NF-B activation. In conclusion, we have demonstrated the
existence of a cell signaling microdomain containing Na,K-ATPase and InsP3R that acts as a
signaling pathway for Ca2 oscillations and activation of NF-B. Our findings represent, in many
aspects, a novel mechanism for generation of Ca2 signaling where an ion pump, Na,K-ATPase, also
functions as a signal transducer. The physiological importance of slow Ca2 oscillations is generally
recognized, yet few biological generators of this signal have been identified previously.
(actuator) (37). A recent study (28) suggests that the NH2 terminus may act as an auto-regulatory
domain, modulating E1/E2 conformational transition. In El conformation, the NH2 terminus is in
association with the first cytoplasmic loop of Na,K-ATPase. Transition from E1 to E2 conformation
may release the NH2 terminus from its interaction with the first cytoplasmic loop of Na,KATPase,
thus making the NH2 terminus available for interaction either with the InsP3R directly or with a
protein bridging between Na,K-ATPase and InsP3R. Such an effect could explain why truncation of
the first 32 amino acids of the NH2 terminus will prevent ouabain-induced Ca2 oscillations. An
alternative explanation is that NH2-terminal truncation has displaced the E1 3 E2 conformation of
Na,K-ATPase in favor of E1 (8, 38). The concept that Na,K-ATPase may, in addition to its function
as an ion pump, also act as a signal transducer, is now rapidly evolving (2-5). Most previous studies
on the signaling role of Na,K-ATPase have been carried out on cardiac myocytes (for a review, see
Ref. 39). In these cells, activation of Src kinase was found to be the primary event in the signaling
cascade initiated by the ouabain/Na,K-ATPase complex, and downstream effects included
phosphorylation of epidermal growth factor receptor and activation of the mitogen-activated protein
kinase pathway. Since cardiac myocytes exhibit spontaneous Ca2 sparks of high frequency, it has not
yet been possible to establish whether Na,K-ATPase/InsP3R-triggered Ca2 oscillations also occur in
these cells. Another model where Na,K-ATPase-mediated Ca2 signaling is dependent on a close
proximity between the plasma membrane and ER Ca2 stores has been presented previously (40). In
this model, Na,KATPase 2- and 3-subunits modulate Ca2 release from ER via local changes in
intracellular Na concentration. Our study was performed on cells expressing only the 1-subunit of
Na,KATPase, and the results from the protocols with the truncated NKA1.M32 imply that increased
intracellular Na concentration was, in our model, not the main cause of ouabain-induced Ca2
oscillations. Furthermore, we previously established that increasing intracellular Na, by lowering
extracellular K to the same extent as 250 M ouabain, is not sufficient to elicit a Ca2 oscillatory
response (2). Ca2 oscillations trigger such universal processes as fertilization, cell division, cell
differentiation, and apoptosis (41). The specificity of this signal is encoded by the frequency and/or
the amplitude of the Ca2 oscillations (42). In the case of G-proteincoupled receptors, Ca2 oscillations
generally have a periodicity of seconds (30, 43). The highly regular ouabain/Na,KATPase-induced
Ca2 oscillations displayed a longer periodicity in the minute range and should thereby provide a
mechanism to ensure a different kind of physiological readout. Endogenous ouabain levels are high
during pregnancy and in the perinatal period (44, 45). Thus, it is of great physiological interest that
the ouabain-induced slow Ca2 oscillations were found to activate NF-B. This pluripotent
transcription factor activates genes modulating cell proliferation, apoptosis, and development as well
as responses of the immune system (46). Cells expressing GFP-NKA1.M32 did not respond to
ouabain with Ca2 oscillations. In these cells, ouabain did not activate NF-B. These findings support
the physiological significance of a signaling microdomain containing Na,K-ATPase 1-subunit and
InsP3R as a trigger mechanism for NF-B activation. In conclusion, we have demonstrated the
existence of a cell signaling microdomain containing Na,K-ATPase and InsP3R that acts as a
signaling pathway for Ca2 oscillations and activation of NF-B. Our findings represent, in many
aspects, a novel mechanism for generation of Ca2 signaling where an ion pump, Na,K-ATPase, also
functions as a signal transducer. The physiological importance of slow Ca2 oscillations is generally
recognized, yet few biological generators of this signal have been identified previously.
Transcribed Image Text:Cell Signaling Microdomain with Na,K-ATPase and Inositol 1,4,5-
Trisphosphate Receptor Generates Calcium Oscillations
It is now generally agreed that many, if not most, important processes in the cell are
controlled by proteins aggregated together in complexes (21, 22, 31-33). The assembly of complexes
that contain a receptor and components of signal machinery provides the cell with a highly selective
means to engage a specific signaling pathway. The finding that ligandbound Na,K-ATPase assembles
with InsP3R and that this assembly can give rise to intracellular Ca2 oscillations with a constant
periodicity in the minute range represents a novel principle for such a protein complex. Based on our
combined results, we suggest that the ouabaininduced Ca2 oscillation and signal-transducing
function of Na,K-ATPase is made possible by the local organization of Na,K-ATPase and InsP3R
into a spatially organized functional microdomain that links the plasma membrane to intracellular ER
Ca2 stores. A signaling microdomain can function without a diffusible messenger provided that the
transducer and the effector are in such proximity that they can communicate via protein-protein
interaction, either directly or via one or more interacting/scaffolding proteins. Molecular strategies
using the InsP3 sponge and pharmacological studies using the PLC inhibitor, U73122, indicate that
the ouabain-induced Ca2 oscillatory response may be elicited via an InsP3-independent mechanism
of InsP3R activation. Significant energy transfer between the donor and acceptor complexes, GFP-
NKA1 and anti-InsP3R-anti-mouse IgG-Cy3, indicate a distance of less than 12 nm (16). It is well
established that the ER is juxtaposed to the plasma membrane (34). FRET was recorded in a region
of the plasma membrane, and the result is clearly compatible with previous electron microscopy
studies showing that the distance between the plasma membrane, where Na,K-ATPase is located, and
the membrane of ER, where the InsP3R is located, can be as short as 10 nm (35). It should be noted,
however, that FRET is recorded between the GFP-labeled Na,K-ATPase and Cy3-labeled goat-anti-
mouse IgG antibody that binds to the InsP3R antibody. Taking the size of the antibodies and the GFP
molecule into account, the maximal distance between Na,K-ATPase and InsP3R could be somewhat
larger than the working distance for FRET fluorophores, GFP and Cy3. Hence, the FRET results do
not rule out the possibility that Na,K-ATPase and InsP3R may interact via a scaffolding protein. The
observed increase in FRET between Na,KATPase and InsP3R following ouabain exposure and the
loss of FRET upon CytD treatment indicate that ouabain-induced Ca2 oscillations were dependent on
a dynamic physical association between Na,K-ATPase and InsP3R and therefore add further
evidence to the concept that the Ca2 signal arises from a signaling microdomain containing Na,K-
ATPase and InsP3R. The question remains as to how the localization of Na,KATPase and InsP3R is
controlled. As evidenced by our FRET and co-immunoprecipitation studies using CytD, an intact
cytoskeleton is required for the physical association between Na,K-ATPase and InsP3R and ouabain-
induced Ca2 signaling. This suggests that actin, or cytoskeletal proteins associated with actin, will
stabilize the Na,K-ATPase/InsP3R complex by physical tether cross-linking. Ankyrins, a family of
adaptor proteins believed to participate in the organization of proteins into specialized regions in the
plasma membrane and ER Ca2 stores, represent an interesting putative mechanistic partner for
orchestrating Na,K-ATPase and InsP3R proximity (25). The complete framework of adaptor and
scaffolding proteins that may be involved in facilitating the structure and signal-transducing function
of this microdomain is still uncertain, but our results also do not preclude the necessity of a direct
physical interaction between Na,K-ATPase and InsP3R in this event. The activation of InsP3R is
likely due to an allosteric effect of ouabain on Na,K-ATPase. Na,K-ATPase is a P-type ATPase that
can exist in distinct E1 and E2 conformational states that are at least partially determined by
intramolecular interactions between the NH2 terminus and cytoplasmic loops of the enzyme.
Ouabain binds to the E2 conformation of the Na,K-ATPase and causes a robust shift in the E1/E2
poise toward E2 forms (36). Information derived from the crystal structure of another P-type
ATPase, the SERCA pump, suggests that the E1 3 E2 state transition is accompanied by significant
Trisphosphate Receptor Generates Calcium Oscillations
It is now generally agreed that many, if not most, important processes in the cell are
controlled by proteins aggregated together in complexes (21, 22, 31-33). The assembly of complexes
that contain a receptor and components of signal machinery provides the cell with a highly selective
means to engage a specific signaling pathway. The finding that ligandbound Na,K-ATPase assembles
with InsP3R and that this assembly can give rise to intracellular Ca2 oscillations with a constant
periodicity in the minute range represents a novel principle for such a protein complex. Based on our
combined results, we suggest that the ouabaininduced Ca2 oscillation and signal-transducing
function of Na,K-ATPase is made possible by the local organization of Na,K-ATPase and InsP3R
into a spatially organized functional microdomain that links the plasma membrane to intracellular ER
Ca2 stores. A signaling microdomain can function without a diffusible messenger provided that the
transducer and the effector are in such proximity that they can communicate via protein-protein
interaction, either directly or via one or more interacting/scaffolding proteins. Molecular strategies
using the InsP3 sponge and pharmacological studies using the PLC inhibitor, U73122, indicate that
the ouabain-induced Ca2 oscillatory response may be elicited via an InsP3-independent mechanism
of InsP3R activation. Significant energy transfer between the donor and acceptor complexes, GFP-
NKA1 and anti-InsP3R-anti-mouse IgG-Cy3, indicate a distance of less than 12 nm (16). It is well
established that the ER is juxtaposed to the plasma membrane (34). FRET was recorded in a region
of the plasma membrane, and the result is clearly compatible with previous electron microscopy
studies showing that the distance between the plasma membrane, where Na,K-ATPase is located, and
the membrane of ER, where the InsP3R is located, can be as short as 10 nm (35). It should be noted,
however, that FRET is recorded between the GFP-labeled Na,K-ATPase and Cy3-labeled goat-anti-
mouse IgG antibody that binds to the InsP3R antibody. Taking the size of the antibodies and the GFP
molecule into account, the maximal distance between Na,K-ATPase and InsP3R could be somewhat
larger than the working distance for FRET fluorophores, GFP and Cy3. Hence, the FRET results do
not rule out the possibility that Na,K-ATPase and InsP3R may interact via a scaffolding protein. The
observed increase in FRET between Na,KATPase and InsP3R following ouabain exposure and the
loss of FRET upon CytD treatment indicate that ouabain-induced Ca2 oscillations were dependent on
a dynamic physical association between Na,K-ATPase and InsP3R and therefore add further
evidence to the concept that the Ca2 signal arises from a signaling microdomain containing Na,K-
ATPase and InsP3R. The question remains as to how the localization of Na,KATPase and InsP3R is
controlled. As evidenced by our FRET and co-immunoprecipitation studies using CytD, an intact
cytoskeleton is required for the physical association between Na,K-ATPase and InsP3R and ouabain-
induced Ca2 signaling. This suggests that actin, or cytoskeletal proteins associated with actin, will
stabilize the Na,K-ATPase/InsP3R complex by physical tether cross-linking. Ankyrins, a family of
adaptor proteins believed to participate in the organization of proteins into specialized regions in the
plasma membrane and ER Ca2 stores, represent an interesting putative mechanistic partner for
orchestrating Na,K-ATPase and InsP3R proximity (25). The complete framework of adaptor and
scaffolding proteins that may be involved in facilitating the structure and signal-transducing function
of this microdomain is still uncertain, but our results also do not preclude the necessity of a direct
physical interaction between Na,K-ATPase and InsP3R in this event. The activation of InsP3R is
likely due to an allosteric effect of ouabain on Na,K-ATPase. Na,K-ATPase is a P-type ATPase that
can exist in distinct E1 and E2 conformational states that are at least partially determined by
intramolecular interactions between the NH2 terminus and cytoplasmic loops of the enzyme.
Ouabain binds to the E2 conformation of the Na,K-ATPase and causes a robust shift in the E1/E2
poise toward E2 forms (36). Information derived from the crystal structure of another P-type
ATPase, the SERCA pump, suggests that the E1 3 E2 state transition is accompanied by significant
