NHE3 is known to be partially active under basal conditions (Donowitz et al., 1985
; Donowitz and Welch, 1986
; Levine et al., 1995
). This characteristic of NHE3 has been identified in every cell system in which it has been studied, in intact intestine, in polarized epithelial cell models, and in fibroblasts. However, the molecular mechanism that maintains this partial activation has not been identified. This study identified that CK2 stimulates NHE3 activity under basal conditions. CK2 binds to the NHE3 C terminus and stimulates NHE3 activity by phosphorylating the NHE3 C terminus at a single site (S719
) that is different from the CK2 binding site. This is an example of the NHE3 C terminus acting as a scaffold for proteins that regulate it. Being set at a partially activated state allows NHE3 to be both inhibited, as occurs during the early phases of digestion, and further stimulated, as occurs in the later stages of digestion. This is in contrast to other transporters which are also regulated by trafficking, such as GLUT4, which are almost entirely intracellular under basal conditions and which traffic to the surface with stimuli, such as insulin, which leads to much greater-fold stimulation of both activity and percentage of increase in amount in the plasma membrane, compared with NHE3 (Pessin et al., 1999
; Watson and Pessin, 2001
CK2 phosphorylation of NHE3 regulates exchanger activity by increasing the percentage of total NHE3 on the plasma membrane by affecting its trafficking. CK2 inhibitors reduced NHE3 activity by ~40% and reduced the percent NHE3 on the plasma membrane by ~50%, consistent with the effects being largely by affecting trafficking. The CK2 effects on trafficking consist of increased endocytic recycling (exocytosis) with no effect in rate of endocytosis or total NHE3 expression. In addition, CK2 phosphorylation of NHE3 was necessary for normal delivery of newly synthesized NHE3 to the plasma membrane. Quantitative effects were somewhat different studying the S719A mutant, which reduced NHE3 activity by ~70% while reducing plasma membrane amount NHE3 by ~60%. Possibilities for the difference between CK2 drug inhibitors and mutation of S719 include that S719A affects NHE3 activity by a mechanism in addition to serving as the CK2 phosphorylation site or that chronic effects of the S719A mutation lead to further secondary changes that do not necessarily directly involve CK2.
The search for unrecognized phosphorylation sites of NHE3 was based on our belief that in spite of NHE3 phosphorylation sites having been identified for cAMP and SGK1, it was likely that there were additional sites present (Donowitz and Li, 2007
). This was because, under basal conditions, the S/T phosphatase inhibitor okadaic acid stimulated NHE3 (Levine et al., 1995
), implying a role for NHE3 phosphorylation in stimulating NHE3, which cAMP inhibits. In addition to the opposite effects on NHE3 activity of phosphorylation by CK2 versus cAMP, another important difference of the effects of CK2 versus PKA and SGK1, is that the latter are brought into the NHE3 complex by binding NHERF1 (PKAII) and/or NHERF2 (PKAII, SGK1), whereas CK2 is brought into the complex by directly binding NHE3.
A role in NHE3 regulation for CK2 was not surprising. It is a ubiquitous kinase with at least the widely quoted 300 recognized substrates, including all classes of proteins that are found in most cellular compartments (Litchfield, 2003
; Olsten and Litchfield, 2004
; Meggio and Pinna, 2005
). Although nuclear proteins seem to be the most affected (Faust and Montenarh, 2000
), CK2 regulates multiple transport proteins in many cell compartments ().
Transporters and channels regulated by CK2
CK2 is a heterotetramer consisting of two catalytic (α, α1
) and two physically joined regulatory β subunits (Faust and Montenarh, 2000
; Filhol et al., 2004
). There are examples of both the α and β subunits binding CK2 substrates, with more examples of β (Litchfield, 2003
; Olsten and Litchfield, 2004
; Bolanos-Garcia et al., 2006
). Moreover, the concept that CK2 binds one protein in a complex and regulates function by phosphorylating another protein in the complex seems to be a common mechanism of action of CK2. For example, CK2 binds KSR1 (kinase suppressor of Ras) and phosphorylates Raf in the multiprotein complex of the extracellular signal-regulated kinase (ERK) cascade (Raf, mitogen-activated protein kinase kinase, ERK) (Allen et al., 2007
Although CK2 generally is thought to function in a constitutive manner, as with the identified NHE3 regulation, there is emerging evidence that it also takes part in regulated functions (Olsten et al., 2005
; Theis-Febvre et al., 2005
; Allen et al., 2007
). Multiple mechanisms have been identified by which CK2 regulates transporter function. In its interactions with the small-conductance Ca2+
activated potassium channels, CK2 is in a complex with the channel, calmodulin (CaM), and PP2A (Allen et al., 2007
). It regulates channel gating by phosphorylating CaM in the multiprotein complex leading to reduction of the Ca2+
sensitivity of the channel (Arrigoni et al., 2004
). Cystic fibrosis transmembrane conductance regulator (CFTR) binds and is regulated by CK2 (Treharne et al., 2007
). CK2 binds wild-type CFTR at approximately aa 505 (very close to the common mutation Phe Δ508) and phosphorylates CFTR at S511
, which seems necessary for cAMP gating of the channel. Binding of CK2 to CFTR requires the presence of the S511
. That is, the substrate phosphorylation site is necessary for nearby CK2 binding. CK2 phosphorylates S1480
of the N
-aspartate (NMDA) receptor, which leads to reduced binding of this transporter to postsynaptic density 95/disc-large/zona occludens domains of PSD95 and SAP102, as well as reduced NMDA surface expression. Thus, there are examples of CK2 phosphorylating transporters and also binding to and phosphorylating the same part of the protein (CFTR). However, unique aspects of transporter regulation by CK2 demonstrated for NHE3 include regulation of basal transport function, contribution to trafficking with effects on exocytosis and delivery of newly synthesized NHE3 to the plasma membrane, and binding to one site but phosphorylating a separate domain in the protein.
We showed here that CK2 phosphorylation of NHE3-S719 stimulates NHE3 activity. Intriguingly, we demonstrated previously that NHE3 truncated at aa 690 (NHE3-690), in which S719
was also removed, exhibits a threefold increase
in activity due to a threefold increase in the amount of NHE3 at the cell surface. If S719
were required for the basal stimulation of NHE3, removal of S719
would have been expected to decrease the basal NHE3 activity. Why did we observe an increase of activity in NHE3-690 instead? We previously identified two endocytosis domains in the C terminus of NHE3: one located at aa 690-756 (which includes S719
) and the other at aa 757-832 (Akhter et al., 2000
). Regulation of WT NHE3 is primarily through trafficking, which is dictated by the balance or interplay between exocytosis (via mechanisms such as CK2 mediated S719
phosphorylation) and endocytosis (via endocytic signals as mentioned above) (Donowitz and Li, 2007
). These results indicate that NHE3 aa, which normally stimulate NHE3 activity (S719
), are interspersed with domains that normally inhibit NHE3 (two endocytosis signals) and show that there is intermixing of stimulatory and inhibitory domains in the NHE3 C terminus. How these interspersed signals functionally interact will be important to understand in the future.
In addition to data with mutation of the NHE3 CK2 phosphorylation site S719
, elucidation of the functional role of CK2 phosphorylation of NHE3 was partially based on study of two pharmacologic inhibitors with high specificity for CK2. DMAT has an IC50
value of 0.14 μM for CK2, with no effect on CK1 at concentrations up to 100 μM (at least 700× less sensitive), whereas TBB has an IC50
value of 0.5 μM for CK2 but inhibits CK1, with an IC50
value of 25 μM (50× less sensitive) (Sarno et al., 2002
; Ruzzene et al., 2002
; Pagano et al., 2004
). Although there are several additional putative CK2 phosphorylation sites in the NHE3 C terminus, the fact that there was no significant effect on NHE3-S719 activity of DMAT at up to 100 μM was consistent with there being only a single NHE3 CK2 phosphorylation site that is linked to regulation of basal NHE3 activity. In contrast, the fact that TBB at high concentrations inhibited NHE3-S719A suggests that a kinase in addition to CK2 contributes to basal NHE3 activity. We suggest that, given that CK1 is inhibited by high concentrations of TBB and that the NHE3 C terminus contains several putative CK1 phosphorylation sequences, it is likely that CK1 also contributes to basal NHE3 activity.
This study identified a newly recognized phosphorylation site in the NHE3 C terminus and showed that it was phosphorylated by CK2 and contributed to setting basal NHE3 activity by increasing the amount of NHE3 on the plasma membrane. Moreover, we identified the specific aspects of NHE3 trafficking that were CK2 dependent, which included exocytosis and delivery of newly synthesized NHE3. In contrast, total NHE3 expression, endocytosis and plasma membrane retention were not CK2 dependent. These studies indicate that the NHE3 C terminus not only acts as a scaffold for some of its regulatory proteins but also there seems to be organization of these proteins (in this case, CK2 binding at one site and phosphorylation at another site) along the C terminus to control NHE3 activity.