The turnover in living cells (Lauf and Joiner 1978

The
present work addresses still unknown details of the cytosolic internalization
of what it is called here, NORC, which is an acronym for the binding and
complex (C) formation of CTS, particularly of ouabain (O), with the NKA (N)
receptor (R). Although not defined as in this work, NORC formation without its
internalization has been known for more than seven decades to precede the
principal and first pharmacological action of CTS to cause inotropy by
inverting the Na/K gradients and stimulating reversal of the Na/Calcium
exchanger leading to increasing cytosolic Ca ions and strengthening of muscle
contraction (modality A) (Nishio et al., 2002, Mohammadi et al., 2003). During
the last 3 decades, NORC formation without internalization was recognized to elicit
signal cascades comprised of tyrosine (Y) kinases starting with the src
tyrosine kinase (Modality B) (Cui and Xie, 2017).PL1 
Subsequent ERK1/2 and MAPK activation are followed by downstream
transcriptional effects.  Activation of
PI3K (Modality C) with subsequent mobilization of cytosolic Ca ions through the
IP3R which in turn augment mitochondrial metabolism of ATP and ROS formation,
again effect transcriptional upregulation of genes important for cellular
survival (Liu et al., 2004, Aperia et al., 2016). However, that NORC, by
cytosolic internalization (modality D), would constitute another mechanism of
cell signaling, after its endosomal and lysosomal processing, is of much more
recent interest, although several publications have suggested this possibility
(Aydemir-Koksoy et al., 2001).

            As it is always in science, advances
in technology facilitated to study above listed 4 modalities, consecutively.
Thus, in modality A, it was the detection of Na + K-dependent

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ATP hydrolysis
by the ouabain-sensitive NKA (Skou et al. 1992, Post R.L. et al. 1972),
cytosolic ionic changes by atomic absorption spectroscopy (Habermann, Chhatwal,
1981), and the tritium labeling of ouabain to assess the NKA pump turnover in
living cells (Lauf and Joiner 1978 a, b), all of which were instrumental
techniques. In modality B and C, molecular biology and protein techniques
enabled detection of src and PI3K association with NKA and the consequent gene
upregulation (Nissen et al., 2012, Pedemonte et al., 2005). Modality D,
neglected for a while since seen first by immunocytochemistry, required
additional special tools such as BODIPY labeling of ouabain, the availability
of confocal microscopy and Förster resonance energy transfer (FRET) analysis (Sekar
et al. 2003).

            One of the first questions to be
solved was that BODIPY fluorescent ouabain (BFO) was transferred as part of
NORC and not simply due to diffusion of the lipophilic compound, either binding
as contamination to the plasma membrane or after a glycosidase-mediated
hydrolysis of the chromophore from ouabain. BFO was detected as part of NORC
within the membrane and cytosol by immune-colocalization of live-staining with
the green fluorescent BFO and the red fluorophore CY3 on the secondary antibody
against the NKA specific antibody, at least 45 min after incubation of B3 cells
with BFO and successive immunochemical staining as shown in Figure 3.3.  Thus, our data resolved the question arising
from the work of Alonso et al 2013: BFO is part of NORC that reaches the
cytosol. An independent approach to demonstration of cytosolic NORC was
provided by dark field amplified microscopy with a CytoViva equipment (not
shown), but this study did not use secondary immune-staining with anti-?1 NKA
antibodies.  However, work on this aspect
is in progress.

            Several proteins known or proposed,
in part based on sequence alignments, to interact with NKA were caveolin-1
(Dong et al., 2011, Yosef et al., 2016, Chakraborti et al., 2015), BcLXL (Lauf
et al., 2015, Aperia et al., 2016), tubulin, early endosomal antigen (EEA1) and
LAMP-1, the lysosomal associated membrane protein 1.

Western
Blot analysis showed the presence of these proteins (Figure 3.2, Figure 3.3).
Furthermore, B3 cells have only the ?1 catalytic subunit (Figure 3.3.) since
the genes for the ?2 and ?3 NKA subunits were absent (Figure 3.1). Thus, B3
cells are typical epithelial cells possessing, always and only, the ?1 subunit
which in all studies published thus far, is tightly linked with the
CTS-activated src-EGFR-ERK1/2 signaling pathway (Cui and Xie, 2017). The
?1subunit is generally associated with the ?1 glycosylated subunit. However,
although transcribed, the Western Blot (Figure 3.3) failed to detect this
protein, instead, the ?1 associates with a ?3 subunit is possible, however, this
interaction has not been further elucidated in this work.

         The B3 cell line is an SV40-immortallized
cell line, where the p53 levels are low or absent (Andley et al., 1994) and
thus apoptosis is minimized, also a hallmark of tumor cells. It is therefore
interesting that B3 cells lack the 27 kDa BclXL with its full set of BH-1, -2, -3,
and -4 motifs, and instead, possess the smaller 19 kDA molecular weight version
of BclXL/S that lack the BH1 motif (Chang et al. 1999), and therefore become a
pro-apoptotic protein carrying the reactive BH-3 hair loop pin that is binding
and neutralizing the fully pro-survival proteins Bcl-2 and BclXL. Since the NKA
?1 subunit has been proposed to contain a BH1-like sequence of 13 amino acids
in its N-terminus, there should be binding of BclXs BH-3 to the former. Indeed,
as Figure 3.5 showed, there was clear colocalization between the CY3-red
stained ?1 NKA subunit and the green fluorescence of anti-BclXL. Since in B3
cells, the classic protein mass staining of 27 kDa is replaced by that of a 19
kDa protein, one must assume that both BclXL and BclXs share a common antigenic
epitope against which anti-BclXL is directed. Given the fact that BclXs is a BH-3
motif only containing peptide, the data suggest that the BclXL and hence BclXs
interactions must occur within the shallow BH1-like domain of the N-terminus of
NKA (a.a. 59-71, see Lauf et al., 2013, and Lauf et al., 2015). It is
interesting that the putative immune colocalization between NKA’s N-terminal BH-1
like domain and BclXs’ BH-3 domain is unaffected by the presence of unlabeled
ouabain from 10-9 to 5x 10-3 M. The N terminal domain
rotates by 120o during the catalytic cycle of NKA to protect the asp
(D)369 ATP-mediated phosphorylation and K-dependent dephosphorylation by in
line hydrolysis. The data indicate that the NKAS-BclXL(s) protein-protein
interaction may not affect the canonical cycle of NKA. However, this conclusion
needs to be functionally tested through ion fluxes in cultures where all cells
show this NKA-BclXL(s) interaction. Indeed, chelerythrine, a BH-3 mimetic,
inhibits the NKA pump fluxes (Lauf et al. 2013), perhaps by a mechanism
proposed here for BclXL(s) that may affect the transit of 2KE2 to 3NaE1~P in
the canonical model.

            Bcl-2 proteins affect the kinetics
of ?-tubulin polymerization (Knipling & Wolf, 2006), and microtubule (MT)
formation has been linked to NKA because a 16-amino acid long peptide in the
C-terminal residues 934-960 interacts with tubulin in the presence of
Tricostatin, an inhibitor of protein deacetylases, i.e. when tubulin is
acetylated (Zampar et al., 2009). Thus, it was only logical to postulate that
part of the intracellular escalator mechanism transporting NORC into endosomes
and lysosomes might be acetylated tubulin. Figure 3.8 shows that indeed there
was immune histochemical evidence for colocalization of NKA with ? tubulin in
some cells but not in others. This effect was amplified when the external
ouabain concentrations was raised to 1 µM. However, in the presence of
Tricostatin and ouabain there was no ?-tubulin-NKA interaction. This data suggests
that it may be the tubulin/NKA protein/protein interaction that is
ouabain-sensitive and accelerated, rather than its acetylated form as proposed
by Zampa et al. (2009). It should be also noted here, that exposure of B3 cells
to ouabain alone increased their cell size (Figure 3.4) commensurate with Na
and water entry into the cells and possibly ? tubulin depolymerization. It is
unknown if such a process drives tubulin-NKA interaction and redistribution as
observed in this work (Figure.3.3, Figure 3.8). There is evidence that MTs
extend from the plasmalemma to mitochondria. Although not tested for tubulin,
Alonso et al., (2013) surmised from their BFO internalization data that, if
proven to be bound to NKA, the latter would associate in the cytosol close to
mitochondria.

Work
from Xie’s group has shown that NKA associates with caveolin-1 (Liu et al.
2005). We confirmed this finding (Figure 3.6). Unless caveolin-1 participates
through tubulin in the cytosolic escalator mechanism, our finding thus constitutes
mainly a control experiment to demonstrate colocalization. However, the
sub-plasmalemmal localization of NORC or NKA with tubulin suggests that the
latter provides the scaffold for the descent of NKA into the endo- and
lysosomal organelles. Our data that NKA colocalizes with EEA1 and LAMP1 are
commensurate with the finding by Karlish and collaborators (2014) that a
YFP-tagged NKA bioconstruct could be traced to early and late endosomes, and to
lysosomes. Interestingly, inconsistent with conventional knowledge, endosomes
in B3 cells must be positioned below the plasma membrane as the yellow/orange
overlap fluorescence indicated.

Conclusion

Our
studies have established:  1). B-3 cells
possess the ?1NKA
subunit. Whether it associates to a functioning unit with ?1
or ?3
is still to be shown. Subsequent to binding of ouabain to the ?1-NKA
receptor, the binary complex NORC is translocated across the plasma membrane as
BFO and antibody labeled NKA colocalized into the cell’s cytosol. 2).
Putatively, NORC interacts with BcLXL/S, and with (non-acetylated) tubulin in an
ouabain concentration-dependent manner. 3). Microtubule (MT) acetylation (by
means of TSA inhibition of the deacetylase) appears to interfere with colocalization
of NKA and MT in the presence of ouabain. Data are not yet available whether
this quaternary complex might contain also caveolin-1. 4). Although we have not
established the time course of events, and other details are missing, it
appears plausible that the NKA without ouabain reaches the endosomes, as
overlap was seen with EEA1. 5). Whether or not NORC does the same for recycling
purpose remains to be seen, more likely NORC associates with late endosomes and
lysosomes, because its colocalization with LAMP1 was ouabain concentration-dependent.
At this point, no further biochemical data are available to further substantiate
these claims and to surmise, NORC may be broken down to its unitary components.
It is projected that ouabain most likely exits in its de-glycosylated form from
these organelles and finds its way to nuclear receptor proteins such as SRC1
and 3 (Wang, O’Malley et al., 2014). 
Ouabain’s interaction with nuclear receptors would then exert its
transcriptional upregulation of NKA as well as pro- and anti-apoptotic
proteins. Work to support this projection is in progress. Figure 4.1 is an
attempt to generate a simplistic model of the cell biology of NORC’s descent
into the cytosol presented in this work.