Regulation of ABC Transporter Function Via Phosphorylation by Protein Kinases

Abstract

ATP-binding cassette (ABC) transporters are multispanning membrane proteins that utilize ATP to move a broad range of substrates across cellular membranes. ABC transporters are involved in a number of human disorders and diseases [1]. Overexpression of a subset of the transporters has been closely linked to multidrug resistance in both bacteria and viruses and in cancer. A poorly understood and important aspect of ABC transporter biology is the role of phosphorylation as a mechanism to regulate transporter function. In this review, we summarize the current literature addressing the role of phosphorylation in regulating ABC transporter function. A comprehensive list of all the phosphorylation sites that have been identified for the human ABC transporters is presented, and we discuss the role of individual kinases in regulating transporter function. We address the potential pitfalls and difficulties associated with identifying phosphorylation sites and the corresponding kinase(s), and we discuss novel techniques that may circumvent these problems. We conclude by providing a brief perspective on studying ABC transporter phosphorylation.

1. INTRODUCTION

ATP-Binding Cassette (ABC) transporters are found in species ranging from bacteria to man [2]. ABC transporters are involved in a variety of human health related problems and diseases (Table 1). The transporters are found in a number of cellular organelles, including the mitochondria, vacuole/lysosome, and plasma membrane, and are responsible for the transport of chemical compounds across cellular membranes in a nucleotide dependent manner [1–4]. The ABC transporters have been divided into 5 subfamilies according to sequence similarity of their nucleotide binding domains (NBDs): ABCA-ABCD and ABCG (ABCE and ABCF are not transporters, have no membrane spans, and are localized to the cytosol) [1,3]. In general, the ABC transporters structurally consist of an “ABC Core” which contains two membrane spanning domains (MSDs or TMDs) and two NBDs that are connected by intracellular loops (Fig. 1) [1,2]. However, transporters can exist as heterodimers of “Half” transporters or “Long” ABC transporters, which contain an additional N-terminal extension (NTE) (Fig. 1) [1,4–6].

Table 1.

ABC Transporter-Associated Diseases

Disease	Transporter(s)	Reference
Tangier disease	ABCA1	[132]
Stargardt disease	ABCA4	[133]
Immune deficiency	ABCB2 and ABCB3	[134, 135]
Progressive familial intrahepatic cholestasis (PFIC)	ABCB4 and ABCB11	[136]
Sideroblastic anemia and ataxia	ABCB7	[137]
Dubin Johnson Syndrome (DJS)	ABCC2	[138]
Pseudoxanthoma Elasticum (PXE)	ABCC6	[139]
Cystic fibrosis	ABCC7	[140]
Persistent hypoglycemia of infancy	ABCC8 and ABCC9	[141]
Adrenoleukodystrophy	ABCD1	[142]
Sitosterolemia	ABCG5 and ABCG8	[143]
Cancer	ABCB1, ABCC1, and ABCG2	[1, 55, 144–146]

Fig. 1. Predicted topology of ATP-Binding Cassette (ABC) transporters.

(A) In general, ABC transporters are structurally characterized as having an ABC “Core” containing two membrane spanning domains (MSDs), also called transmembrane spanning domains (TMDs), and two intracellular nucleotide binding domains (NBDs). A number of ABC transporters exist as “Half” transporters which form homo- and/or heterodimers. (B) The ABCC subfamily has N-terminal extension (NTE) in addition to the ABC “Core”, The NTE of “Long” ABCC proteins consists of an extra membrane spanning domain (MSD0, TMD0) and an extra linker domain (L0) (also called cytoplasmic loop 3 (CL3)). In “Short” ABCC proteins, the MSD0 is absent. (C) The linker region between NBD1 and MSD2 of ABCC7 (CFTR) is extensively phosphorylated and is thus referred to as the regulatory domain (R domain). Similar domains are found in other ABC transporters and are referred to here as the “R-like” domain (annotated in Fig. 1A).

Although much is known about the function of the ABC transporters, post-translational regulation of transporter function has remained relatively uncharacterized until recently. A significant problem associated with identifying proteins that regulate ABC transporter function is the detection of interacting proteins. For example, identifying an interacting kinase(s) and the corresponding post-translational modification(s) is difficult [4,7]. The difficulty arises from the fact that ABC transporters are tethered to the membrane but site-specific or truncation mutants often misfold or un- fold and as a result traffic incorrectly [4]. The development and improvement of a number of techniques including the membrane yeast-two hybrid (MYTH) assay and proteomic methods, such as co-immunoprecipitation-affinity chromatography followed by liquid chromatography-mass spectrometry (LC/MS) proteomic analysis, over the last decade has greatly reduced this barrier [4]. Many phosphorylation sites within ABC transporters have been identified as a result of these improvements. In many cases, the corresponding kinase(s) has also been identified [4,7]. This review will focus on regulation of ABC transporter function via phosphorylation, the kinases that are involved, and the current and future technologies that are and will be available to identify new kinases that phosphorylate the ABC transporters as well as their corresponding phosphorylation sites.

2. PHOSPHORYLATION AS A MECHANISM OF ABC TRANSPORTER REGULATION

Phosphorylation is one of the most common mechanisms of post-translational protein regulation in the cell [8]. A broad range of cellular proteins are regulated via kinase-mediated phosphorylation. Phosphorylation occurs under greatly varying conditions in response to multiple stimuli [9,10]. Phosphorylation is critically important for the regulation of a number of cellular processes including transcription, ubiquitination, protein degradation, protein subcellular localization, and, most notably, protein function [10]. The availability of ATP as a donor and the ease of reversibility of phosphorylation are likely the reasons why the cell has so extensively adopted phosphorylation as a mechanism to regulate so many cellular processes [8]. Therefore, it is not surprising that abnormal phosphorylation is associated with many human diseases and conditions (misregulation of a number of kinases is strongly associated with multidrug resistance (MDR) in cancer) [9,10]. Many diseases occur as the result of mutated phosphorylation sites [8]. It is important to note that many toxins and pathogens exert their effects by altering cellular phosphorylation [8]. One such example of a phosphorylation-altering toxin is microcystin, which is effluxed by the green algae that produces it via the mycH ABC transporter. This toxin inhibits Type 1 and 2A protein phosphatases. Importantly, microcystin is a substrate for P-gp (ABCB1) in fish and is believed to regulate the expression and possibly the function of fish P-gp via inhibition of protein phosphatases [11–13].

A critical gap in our understanding of ABC transporter biology is the mechanism by which the transporters are regulated at the post-translational level [4]. A number of studies have identified phosphorylation as a mechanism of transporter regulation. Phosphorylation sites have been identified in members of almost every ABC transporter subfamily from yeast to man. It is reasonable to believe that all the transporters are regulated by phosphorylation and a variety of other post-translational modifications to some extent. Regulation of ABC transporter function by phosphorylation provides cells with a simple, low energy, fast, and efficient way to change transporter function. Other modifications that may regulate ABC transporter function or expression, such as sumoylation or ubiquitination, require more energy and are less efficient. A comprehensive list of phosphorylation sites identified by LC/MS for the human ABC transporters is provided in Table 2. Below we will discuss the role of phosphorylation as a mechanism to regulate the function of each ABC subfamily. We will conclude this review with a prospectus for how modern proteomics will aid in identifying the post-translational regulators and post-translational modifications of the ABC transporter family.

Table 2.

Phosphorylation Sites of the ABC Transporters Identified by MS Analysis of Tryptic Derived Peptides

Transporter	Amino acid	Site	Reference
ABCA1	SERINE	1042	[14,15]
	THREONINE	1242
	THREONINE	1243
	SERINE	1255
	SERINE	2054
ABCA2	SERINE	1327	[147]
	SERINE	1331
	TYROSINE	1694	[148]
	TYROSINE	2178	[149]
	TYROSINE	2186	[148]
	THREONINE	2412	[149]
ABCA3	TYROSINE	1265	[148]
	TYROSINE	1268
	TYROSINE	1349
ABCA4	TYROSINE	400	[148]
ABCA5	TYROSINE	22	[148]
	TYROSINE	58
	TYROSINE	1299	[150]
ABCA6		NONE IDENTIFIED
ABCA7		NONE IDENTIFIED
ABCA8		NONE IDENTIFIED
ABCA9		NONE IDENTIFIED
ABCA10	TYROSINE	307	[151]
	SERINE	568	[152]
ABCA11		NONE IDENTIFIED
ABCA12	TYROSINE	811	[150]
	TYROSINE	814
ABCA13	TYROSINE	1773	[148]
ABCB1	SERINE	644	[149]
	SERINE	658
	SERINE	660
	SERINE	661	[23,24]
	SERINE	667
	SERINE	671
	SERINE	683	[149]
	SERINE	686	[23,24]
ABCB2		NONE IDENTIFIED
ABCB3	THREONINE	458	[149]
	TYROSINE	597	[150]
	TYROSINE	693
ABCB4		NONE IDENTIFIED
ABCB5		NONE IDENTIFIED
ABCB6		NONE IDENTIFIED
ABCB7	TYROSINE	345	[148]
	TYROSINE	356	[148]
ABCB8		NONE IDENTIFIED
ABCB9	SERINE	530	[153]
ABCB10		NONE IDENTIFIED
ABCB11		NONE IDENTIFIED
ABCC1	TYROSINE	490	[148]
	SERINE	915	[154,155]
	SERINE	917	[156]
	SERINE	918	[150]
	SERINE	919	[157,158]
	TYROSINE	920	[148,159,160]
	SERINE	930	[161,162]
	TYROSINE	1508	[160]
ABCC2	SERINE	283	[149,154]
	TYROSINE	616	[157,158]
	THREONINE	873	[149]
	SERINE	878
	TYROSINE	885
	SERINE	900
ABCC3	SERINE	908	[149,163]
	SERINE	911
ABCC4	SERINE	313	[152]
	SERINE	314
	SERINE	601	[164]
	THREONINE	646	[147,149,155]
	TYROSINE	1259	[150]
ABCC5	TYROSINE	10	[160]
	SERINE	505	[149]
	SERINE	509
	THREONINE	513
ABCC6	SERINE	681	[156]
	SERINE	902	[165]
	SERINE	1310	[156]
ABCC7	SERINE	422	[166]
	SERINE	489
	SERINE	511	[86]
	TYROSINE	515	[167]
	SERINE	519	[71]
	SERINE	557
	THREONINE	582	[77]
	THREONINE	604
	SERINE	641	[71]
	SERINE	660	[63,166,168]
	SERINE	670	[71]
	THREONINE	682	[77]
	SERINE	686	[63]
	THREONINE	690	[71]
	SERINE	700	[63,166,168]
	SERINE	707	[77]
	SERINE	712	[166,168]
	SERINE	728	[71]
	SERINE	737	[63,166,168]
	SERINE	742	[71]
	SERINE	753	[168]
	SERINE	756	[71]
	THREONINE	757
	THREONINE	760
	SERINE	768	[63,166,168]
	THREONINE	787	[71]
	THREONINE	788
	SERINE	790	[63]
	SERINE	795	[63,166,168]
	SERINE	813
	THREONINE	1176	[156]
ABCC8	TYROSINE	798	[150]
ABCC9		NONE IDENTIFIED
ABCC10	THREONINE	463	[169]
	SERINE	467
ABCC11	TYROSINE	993	[157,158]
ABCC12		NONE IDENTIFIED
ABCD1	SERINE	733	[149,154,170]
ABCD2		NONE IDENTIFIED
ABCD3	TYROSINE	143	[152]
ABCD4		NONE IDENTIFIED
ABCG1		NONE IDENTIFIED
ABCG2	TYROSINE	362	[105]
ABCG4		NONE IDENTIFIED
ABCG5		NONE IDENTIFIED
ABCG8		NONE IDENTIFIED

3. ABC TRANSPORTER SUBFAMILIES; CONSEQUENCES OF THEIR PHOSPHORYLATION

3.1. ABCA

The ABCA subfamilies of proteins are involved in a variety of cellular functions and are associated with a number of human diseases [1,2]. Most notable is the role of ABCA1 in cholesterol efflux and Tangier disease. ABCA4 is critically important for the efflux of the retinol-AC conjugate from retinal pigment epithelium cells. Mutations in ABCA4 result in Stargardt disease, which is similar to macular degeneration [1,2]. Of these two transporters, ABCA1 has been the most extensively studied. For many years protein kinase A (PKA) was known to play an important role in regulating cholesterol efflux; however, the mechanism for this regulation was unknown. In 2002 PKA-regulated cholesterol efflux was finally shown to be the result of direct phosphorylation of ABCA1[14]. The study identified two PKA phosphorylation sites at serine (Ser)1042 within NBD1 and the homologous site, Ser2054, within NBD2 (it is thought that the NBDs share significant homology due to gene duplication) [14]. Although both sites are PKA phosphorylation sites in vitro, mutation analysis in vivo strongly suggests that Ser2054 is the dominant phosphorylation site [14]. Phosphorylation of Ser2054 is constitutive and required for full transporter activity. Mutation of Ser2054 to alanine (Ala) results in a 50% decrease in ABCA1-mediated cholesterol efflux in vitro [14]. In 2004, Roosbeek et. al. published biochemical evidence for phosphorylation of the CK2 consensus sites at threonine (Thr)1242, Thr1243, and Ser1255 within the “R-like” domain (Fig. 1), a shorter version of the ABCC7 R domain found between NBD1 to MSD2 [15]. Mutation of these sites individually to alanine (Ala) results in a partial loss of protein function [15]. Further, treatment of cells with a CK2 inhibitor resulted in decreased phosphorylation of recombinant NBD1-R1 (from ABCA1), which supports the initial finding that Thr1242, Thr1243, and Ser1255 are phosphorylated directly by CK2 [15].

Importantly, phosphorylation of ABCA1 plays a role in protein stability [16,17]. Martinez et. al. showed that phosphorylation of ABCA1 at Thr1286 and Thr1305, within the “R-like” domain, promotes calpain-mediated ABCA1 degradation [16]. Mutation of both sites to Ala resulted in a 3.1-fold increase in cell surface expression and a 2.3-fold increase in cholesterol efflux as compared to wild type (WT) [16]. Further, Yamauchi et al. provided evidence suggesting that ABCA1 is stabilized through a protein kinase Cα(PKCα)-dependent phosphorylation mechanism [17]. Together, these studies suggest a role for phosphorylation in the regulation of ABCA1 protein activity and stabilization/degradation.

3.2. ABCB

The ABCB subfamily of ABC transporters is a structurally and functionally diverse group of proteins that is conserved in all mammals (reviewed elsewhere) [1,2,18]. Members of the ABCB subfamily have been directly linked to multiple diseases including cholestasis, immune deficiency, and sideroblastic anemia, and MDR in cancer [19]. The most recognized of the ABCB subfamily is probably ABCB1, which is more commonly referred to as MDR1 or p-glycoprotein, followed by ABCB2 and ABCB3, the transporter-associated with antigen presenting proteins (TAPs) [1,3,19].

3.2.1. ABCB1

Overexpression of ABCB1 is strongly associated with the multidrug resistance (MDR) phenotype in a broad range of cancers [1,20]. Although the role of ABCB1 in cancer has been extensively studied, very little is known about the role of ABCB1 in normal cellular metabolism and cell protection from environmental stress. In addition, post-translational regulation of ABCB1 function is poorly understood. Interestingly, a large number of studies have suggested that ABCB1 is phosphorylated in vivo; however, it is still unclear what role phosphorylation plays in regulating ABCB1 function [8,9,20–43]. In 1987, Mellado and Horwitz published the first evidence suggesting that ABCB1 was phosphorylated [38]. Their work showed that phosphorylation of ABCB1 increased when cells were treated with cAMP. Treatment of partially purified ABCB1 with recombinant PKA (i.e., the catalytic subunit) results in increased phosphorylation of ABCB1 in vitro [38]. It is now apparent that ABCB1 is likely phosphorylated by a number of kinases, including PKC and PKA. A number of conflicting studies have been published as to the role of phosphorylation in regulating ABCB1 function and yet no clear consensus has been reached [21–25,29,31–32,36,41,42].

Interestingly, it is important to note that a large number of studies have shown that many of the known inhibitors of PKA, PKC, and many other kinases are both substrates and/or inhibitors of ABCB1 function [44–48]. There is increasing evidence that the same is true for some of the ABCC and ABCG subfamilies of transporters. The role of the kinase inhibitors as substrates and inhibitors of the ABC transporters is extremely important and critical to evaluating the effectiveness of kinase inhibitor use in the clinic [44–48]. Although of extreme importance, the role of kinase inhibitors as substrates and inhibitors of the ABC transporters is not covered in this review. Excellent reviews on this subject can be found elsewhere [44–48].

A number of studies support a role for phosphorylation in the regulation of ABCB1 function [21,22,25,30,31,34,36,39–42]. Overwhelming evidence suggests that PKC is a major player in ABCB1 phosphorylation and regulation [22–25,30–32,37,39,40,42,43]. Ex vivo purification of ABCB1 followed by tryptic digestion and peptide sequencing via Edman degradation identified that human ABCB1 is phosphorylated at putative PKC phosphorylation sites: serine 661, 667, and 671 [24,39]. Supporting these findings, in vitro kinase assays performed on small peptides derived from the “R-like” domain of ABCB1 identified serine 661, 667, and 671 as potential in vivo PKC phosphorylation sites [23,27,28]. PKC-dependent phosphorylation of ABCB1 is stimulated by the PKC activator 12-O-tetradecanoylphorbol-13-acetate (TPA) and okadaic acid [24] and inhibited by sphingosine stereoisomers [40]. Work by Idriss et. al. examining ABCB1 function in purified vesicles from sf9 cells suggests that phosphorylation within the “R-like” domain is specific for PKCα and not PKCε [31]. Further phosphorylation within the “R-like” domain regulates ABCB1 ATPase activity [31,42]. PKCα-mediated phosphorylation appears to regulate ABCB1-dependent efflux of anions, which suggests a role for ABCB1 in Cl⁻ channel regulation [30–32,42–43]. Interestingly, mutation of all the possible PKC phosphorylation sites within the “R-like” domain of ABCB1 does not alter ABCB1 function [29]. However, the results of this study must be taken with caution as the function of the mutants was assessed in yeast and not in mammalian cells [29]. It is not uncommon to find that studies performed in yeast with mammalian homologues differ in their results as compared with the homologous studies carried out in human/mammalian cells [49–51].

In addition to PKC, evidence suggests that PKA and CK2 phosphorylate ABCB1 and regulate ABCB1 function [24,27–28,39]. PKA phosphorylates serine 667, 671, and 683 in human ABCB1 in vitro and in vivo [24,28,39]. These studies suggest that serine 667 and 671 are substrates for both PKC and PKA. CK2 phosphorylates up to five sites of ABCB1 within a small tryptic fragment (631–658) within the “R-like” domain similar to PKC and PKA [27]. Interestingly, GTP can stimulate ABCB1 phosphorylation and the intracellular ATP/GTP ratio has a clear effect on ABCB1 phosphorylation status [33,35]. In conclusion, ABCB1 function is regulated by phosphorylation and this regulation is quite dynamic due to the involvement of multiple kinases and multiple phosphorylation sites [34].

3.2.2. ABCB2 and ABCB3 (TAP1 and TAP2)

ABCB2 and ABCB3 are commonly referred to as the ATP-binding cassette transporters associated with antigen processing or TAPs [1,2]. ABCB2 and ABCB3 are required for the transport of antigenic peptides from the cytosol into the lumen of the ER for the assembly of histocompatibility antigen complex class I [1,2]. Heterodimerization is required for ABCB2 and ABCB3 to carry-out their cellular function [1,2]. Li et al. showed that a 43-kDa kinase can be found associated with the ABCB2/3 complex [52]. ABCB2/3 transport activity is inhibited by phosphorylation [52]. However, with such limited number of studies analyzing the role of phosphorylation on ABCB2/3 function, no significant conclusions can be made.

3.3. ABCC

The ABCC transporters are distinct from other ABC transporters by two unique features: 1) many transport glutathione conjugates (GS-X) or cotransport glutathione (GSH) and a substrate; and 2) the subfamily has an additional N-terminal extension beyond the “ABC Core” (Fig. 1) [2,53–55]. The N-terminal extension comes in two forms, a long form (comprising 5 additional membrane spans and a cytosolic loop) and a short form (comprising a very large N-terminal cytosolic domain of about 300 amino acids) (Fig. 1) [2,53,54]. The ABCC subfamily is one of the largest ABC transporter subfamilies. The ABCCs are involved in a variety of human diseases including pseudoxanthoma elasticum, cystic fibrosis, and MDR. ABCC7, more commonly referred to as the cystic fibrosis conductance regulator (CFTR) [2], is the most characterized of all the ABC transporters with respect to phosphorylation. Phosphorylation of the ABCC subfamily is discussed below.

3.3.1. ABCC2

ABCC2 is the gene that is responsible for Dubin-Johnson Syndrome (DJS) [1,2]. One of the major roles of ABCC2 in humans is thought to be the transport of conjugated bilirubin across the plasma membrane and into the canacular space where it can be removed from the body via hepatobiliary excretion 1,2]. Mutations that render ABCC2 non-functional prohibit the efflux of conjugated bilirubin from the liver. The loss of ABCC2 function or a reduction in transporter function results in the intracellular accumulation of both conjugated bilirubin (glucuronide-bilirubin) and unconjugated bilirubin in the liver [56,57]. Therefore, one mechanism of potential treatment of DJS patients would be to upregulate ABCC2 function, and hence, a critical understanding of ABCC2 regulation is required. To date, only three papers have reported on the posttranslational modification of ABCC2 [58–60]. Of these three reports, only one provides significant evidence to suggest a role for phosphorylation in ABCC2 regulation [60]. Although further investigation is needed, the initial studies described here suggest that patients with Dubin Johnsons Syndrome may benefit from treatment with kinase inhibitors.

3.3.2. ABCC7

ABCC7, or CFTR as it is more commonly known, is the Cl-channel that is responsible for cystic fibrosis when mutated [18,61]. ABCC7 is a short ABCC similar to ABCC4 and 5 (Fig. 1). Mutations in ABCC7, most notably a deletion of phenylalanine (Phe)508, result in misfolding of the protein and retention in the endoplasmic reticulum (ER) [18,61]. Of all the members of the ABC transporter superfamily, ABCC7 regulation via kinase-mediated phosphorylation is the most extensively characterized (see the list of identified phosphorylation sites in Table 2). The first evidence of ABCC7 regulation via phosphorylation was by Tabcharani et. al. and Picciotto et. al. in 1991 [62–64]. Their studies suggested that ABCC7 is regulated by PKA and PKC in response to cyclic AMP (cAMP) [62–64]. It has now been almost 20 years since this initial discovery and only very recent work has begun to shed light on the mechanism by which phosphorylation regulates ABCC7 function. ABCC7 phosphorylation and regulation have been extensively reviewed elsewhere [40,65–71], and thus we will briefly summarize the findings and discuss them in the context of the larger ABC transporter superfamily.

A review of the literature suggests that ABCC7 is extensively phosphorylated throughout the R domain, a cytosolic region of the protein that links the NBD1 to MSD2 see Table 2 and Fig. (1). ABCC7 function appears to be regulated by phosphorylation within NBD1 by PKC [72–74], and by extensive PKA- and PKC-mediated phosphorylation within the R domain [64,72,75–82]. In addition, more recent work suggests that ABCC7 function is regulated to a lesser extent by AMPK- and CK2-mediated phosphorylation within the R domain, and dephosphorylation by protein phosphatases [62,83–87].

The R domain of ABCC7 is rich in putative PKA phosphorylation sites [62–64,66,67,71]. In general, individual or combined mutation of PKA sites results in decreased transporter function [62–64,66,67,69,71,72,76,77,80,88–90]. The stimulatory effect of PKA on ABCC7 via phosphorylation is additive with a maximal induction of ABCC7 transporter function around 3-fold over that of the unphosphorylated protein [66,67,71]. Importantly, detailed analysis of the phosphorylation sites relative to each other suggests that phosphorylation at any one site is directly and indirectly affected by phosphorylation at another phosphorylation site(s) within ABCC7 [66,67,71]. PKA has been shown to require AKAP (Ezrin) to physically interact with ABCC7 and inhibition of this interaction blocks PKA phosphorylation of the R domain [82]. This finding suggests that phosphorylation of ABCC7 by PKA is regulated by AKAP.

Similar to PKA, ABCC7 has multiple PKC consensus sites within the R domain [62–64,66,67,71]. Regulation of ABCC7 function via PKC-mediated phosphorylation within the R domain is influenced by PKA- and PKC-mediated phosphorylation at other, non-R domain localized phosphorylation sites [66,67,71]. Therefore, PKA- and PKC-mediated ABCC7 regulation is extremely complex and involves multiple phosphorylation sites [66,67,71]. In the future, it will be very important to analyze the phosphorylation status of ABCC7 under various cellular conditions.

Recent studies shed new light on the mechanism by which phosphorylation regulates ABCC7 function through the R domain [70,78,91,92]. A combination of NMR, computational, and three dimensional cryomicroscopy studies indicate that phosphorylation of the R domain results in conformational changes that alter the function of ABCC7 [68,70,78,92]. The computational studies suggest that the change afforded by phosphorylation pushes the R domain away from the core and NBDs which results in an increase in the apparent size/radius of the protein [68]. This computational model is further supported by three dimensional cryomicroscopy studies which identified that in the absence of phosphorylation of the R domain, the NBDs and the core are more compact in shape and structure [68,70,92]. The culmination of these findings was recently published by Kanelis et al. In this study, the authors demonstrate by NMR that the unphosphorylated R domain of ABCC7 folds into the NBDs and the core. Phosphorylation of the R domain inhibits these interaction [92]. Together these studies suggest a model in which phosphorylation of the R domain prevents “compacting” of the ABCC7 protein and opens the core to accept ions for transport [92]. These findings provide important insight into the overall role of phosphorylation in the regulation of all ABC transporters. Although the extensive cytosolic loop in ABCC7 that connects NBD1 to MSD0 is not strictly conserved in other ABC transporters, a similar structure is found in every member of the ABC transporter superfamily [2,19].

3.3.3. ABCC8/9

ABCC8 and ABCC9, more commonly called sulfonyl urea receptors 1 and 2 (SUR1 and SUR2), are “Long” ABCC transporters similar to ABCC1 and ABCC2 [18,19,93]. Unlike other members of the ABCC subfamily, ABCC8 and ABCC9 are part of a multiprotein complex containing multiple subunits of the potassium inward rectifier protein, Kir6. 2. This large protein complex forms classical K_ATP channels [94]. Mutations in ABCC8, ABCC9, and Kir6.2 result in familial persistent hyperinsulinemic hypoglycemia (PHIP) [18,19,93,94] and appear to play an important role in cardiomyocyte function [95]. Similar to many other cellular ion channels, the K_ATP channels are highly regulated at multiple levels including post-translationally by phosphorylation [96–102].

Multiple studies suggest that ABCC8 is regulated by phosphorylation. One such study reports that ABCC8 (SUR1) is phosphorylated at Ser1571 in a PKA-dependent manner [96]. Phosphorylation of ABCC8 by PKA regulates the basal state and function of the K_ATP channels, including burst duration and interburst intervals[96]. Interestingly, the same study identified a PKA phosphorylation site at Ser372 in Kir6.2 and demonstrated that this site induces K_ATP channel activity in vivo [96]. This work is supported by Lin et. al. which identified a second phosphorylation site within Kir6.2 at Thr224 [98]. In addition to PKA regulation, ABCC8 may be regulated by PKC [100]. Work by Ribalet et. al. suggests that phosphorylation of both Kir6.2 and ABCC8 is required for proper function of the K_ATP channel [100]. To this end, cells expressing functional K_ATP channels were inhibited by specific PKC inhibitors [100].

Similar to ABCC8, ABCC9 (SUR2) is phosphorylated by PKA [99,101–103] at Thr633, Ser1387, and Ser1465 [99,103]. Phosphorylation of ABCC9 by PKA induces activation of the K_ATP channel [99,103]. This work, together with the studies described above for ABCC8, suggests a role for multisite phosphorylation as a complex mechanism for regulating K_ATP channel activity [99]. A challenge to studying the mechanism by which phosphorylation regulates ABCC8 and ABCC9 is determining how their interaction with Kir6.2 is affected. Therefore, it will be interesting to see if recent advances in phosphoproteomics and structural biology will lead to new insights into the role of phosphorylation in regulating K_ATP channel activity.

3.4. ABCD

The ABCD subfamilies of ABC transporters are located within the membrane of the peroxisome [1,2]. There are four members of this subfamily, ABCD1–4 [1,2]. Mutations within ABCD1 are responsible for the human disease, adrenoleukodystrophy [1,2]. To date, only one study has reported that members of the ABCD subfamily are regulated by phosphorylation [104]. Tanaka et. al. reported that ABCD1 and ABCD3 are phosphorylated in vivo and this phosphorylation appears to alter transporter function [104]. Unlike many of the other ABC transporters, which are phosphorylated by Ser/Thr-specific kinases, ABCD1 and ABCD3 appear to be phosphorylated by a tyrosine kinase [104]. In conclusion, it is reasonable to believe that ABCD1 and ABCD3 are regulated via phosphorylation; however, a considerable amount of work remains before any substantial conclusions can be drawn.

3.5. ABCG

The ABCG subfamily of transporters is an extremely diverse subfamily of transporters comprised of five half transporters (ABCG 1,2,4,5, and 8) [2]. The ABCG subfamily includes ABCG2 (BCRP), which is has been strongly associated with drug resistance in cancer, and ABCG5 and ABCG8, which have been shown to be responsible for sitosterolemia [2]. Similar to the ABCD subfamily, only one report to date has identified phosphorylation as a potential regulatory mechanism in this subfamily [105]. Xie et. al. reported that phosphorylation of ABCG2 is required for proper function [105]. Their work suggests that the kinase Pim-1 phosphorylates ABCG2 at Thr362 and that this phosphorylation modulates dimerization of the ABCG2 molecules, which is a requirement for proper function [105]. Mutation of Thr362 to alanine results in cytoplasmic compartmentalization of ABCG2 thereby inhibiting proper localization at the plasma membrane and protein dimerization [105]. Further work is necessary to determine the overall extent to which ABCG2 is phosphorylated.

4. REGULATION OF NON-MAMMALIAN ABC TRANSPORTERS: A BRIEF OVERVIEW

ABC transporters are found in all organisms from bacteria to man [2,18]. The role of ABC transporters in microorganisms has been associated with the efflux of a number of compounds and ions including salt ions, sugars, lipids, and toxins [106–114]. In bacteria and yeast, ABC transporters are associated with multidrug resistance and increased organism virulence [106–114]. Therefore, a better understanding of ABC transporter biology and biochemistry is of critical importance in order to improve treatment of microorganism-associated diseases and infections. In addition, microorganisms, such as the yeast Saccharomyces cerevisiae, are excellent models for the study of ABC transporter function and regulation [4,115,116]. In recent years, high throughput genomic and protein interaction studies involving yeast ABC transporters have proven extremely useful in identifying new regulatory pathways including those involving phosphorylation [4,115,116]. Recent advances in phosphoproteomics of the whole yeast proteome have been extremely useful in identifying a number of phosphorylation sites within the ABC transporters [110,117–120]. Together, these approaches have provens the utility and importance of yeast and other microorganisms as model systems to characterize ABC transporter function.

To date a number of studies in yeast and bacteria have identified mechanisms by which ABC transporter function is regulated by phosphorylation [121–129]. In yeast, the ABC transporters that have been suggested to be regulated by phosphorylation include Ste6p, Ycf1p, Pdr5p, and Cdr1p [122–127,129]. Phosphorylation of Ste6p, a protein required for yeast mating, appears to play a critical role in regulating protein localization and recycling [122,123]. The activity of Pdr5p is regulated by Sit4p-mediated phosphorylation [126]. Similarly, Ycf1p function is regulated by the yeast casein kinase 2 alpha protein, Cka1p [124]. Phosphorylation has been suggested to play a dual role for Cdr1p [129] by altering the activity and stability of the transporter [129]. Similar studies can be found for bacterial ABC transporters [121,128]. Mycobacterium tuberculosis pathogenicity in mice requires the ABC transporter, Rv1747 [121,128]. Here pathogenicity associated with Rv1747 is regulated, in part, via a Ser/Thr kinase [121,128]. Further insight into the mechanisms regulating ABC transporter phosphorylation in microorganisms may allow for the development of novel kinase inhibitor-based therapeutics for the treatment of microorganismal-associated diseases and infections.

5. PHOSPHOPROTEOMICS: PROSPECTUS WITH RESPECT TO ABC’S

With the increasing development of new phosphopeptide enrichment techniques and the increasing sensitivity of mass spectrometers, the identification of phosphorylation sites on proteins has become quite routine [130]. With the ever increasing number of high quality proteomic cores being established, liquid chromatography-mass spectroscopy (LC/MS) has quickly become the technique of choice [130]. Unlike many of the traditional techniques used to identify phosphorylation of a protein, such as two dimensional gel electrophoresis (2D SDS-PAGE) analysis of [³²P]-labeled protein lysates, LC/MS does not require radioactivity, it is more sensitive, and has the ability to identify multiple phosphorylation sites in multiple samples during a single run. This allows for a direct comparison of the phosphorylation status of a single protein from two separate samples. LC/MS in this manner utilizes a technique called stable isotope labeling of amino acids in tissue culture or SILAC [130].

One of the most important developments in recent years may be the use of immobilized metal affinity chromatography (IMAC) to greatly enrich phosphopeptides from whole-cell trypsinized lysate protein preparations [131]. Currently, ultra high performance liquid chromatography (UHPLC) and nano-HPLC MS/MS analysis in combination with various dissociative MS techniques (ECD and ETD) have increased detection sensitivity well beyond what was capable even a year ago [131]. Improvements in all these areas will increase our ability to detect phosphorylation sites within the ABC transporters, a development that will be essential for understanding the complex regulation of ABC transporter function via multisite phosphorylation.

Although the development of highly sensitive phosphoproteomic detection techniques has vastly improved our ability to identify sites of phosphorylation, it remains extremely difficult to identify phosphorylation sites within membrane bound proteins [7]. As with the study of membrane proteins in general, a great limitation to their biochemical analysis is our ability to enrich them [7]. Therefore, it has become critically important to consider one or multiple enrichment steps [7]. To use LC/MS phosphoproteomics successfully to identify ABC transporter phosphorylation sites, multiple techniques have been used. These techniques include both classical and modern methodologies such as density gradient centrifugation, co-immunoprecipitation (possibly by biotinylation), and purification from cell debris with various detergents [7]. Other techniques that will continue to play an important role in the identification of kinases and kinase-transporter interactions are cross-linking technologies and the modified yeast two hybrid assay (iMYTH). “Cell-shaving” may provide unique insights into the status of the externally oriented loops of the ABC transporters [4,7]. These techniques are extensively reviewed by Cordwell et. al. [7].

In conclusion, advanced tools are available to further address how phosphorylation regulates ABC transporter function. More detailed analysis of this type will provide useful insight into not only the cellular function of ABC transporters but also into how these proteins are regulated. Ultimately, the identification of kinases that regulate transporter function may aid in the development of novel kinase inhibitor-based therapies that will more effectively treat ABC transporter-related diseases.

LIST OF ABBREVIATIONS

ABC

ATP-binding Cassette

BCRP

Breast Cancer Related Protein

CFTR

Cystic Fibrosis Transductance Regulator

IMAC

Ion Metal Affinity Chromatography

LC/MS

Liquid Chromatography/Mass Spectrometry

MRP

Multidrug Resistance-associated Protein

P-gp

P-Glycoprotein

TAP

Tandem Affinity Purification

UHPLC

Ultra High Performance Liquid Chromatography

ECD

Electron Capture Dissociation

ETD

Electron Transfer Dissociation