Mass spectroscopy identifies the splicing-associated proteins, PSF, hnRNP H3, hnRNP A2/B1, and TLS/FUS as interacting partners of the ZNF198 protein associated with rearrangement in myeloproliferative disease

ZNF198 is fused with FGFR1 in an atypical myeloproliferative disease that results in constitutive activation of the kinase domain and mislocalization to the cytoplasm. We have used immunoprecipitation of a GFP-tagged ZNF198 combined with MALDI-TOF mass spectroscopy to identify interacting proteins. P splicing factor (PSF) was identified as one of the proteins and this interaction was confirmed by Western blotting. Other proteins identified were the spliceosomal components hnRNP A2/B1, hnRNP H3, and TLS/FUS. PSF is also known to interact with PTB, another member of the hnRNP family of proteins, and we further demonstrated that PTB interacts with ZNF198. The interaction between TLS/FUS and ZNF198 was confirmed using Western blot analysis. In 293 cells expressing the ZNF198/FGFR1 fusion protein, neither PSF nor PTB binds to the fusion protein, possibly because of their differential localization in the cell.     

Critical role of STAT5 activation in transformation mediated by ZNF198-FGFR1

The 8p11 myeloproliferative syndrome is an aggressive disorder caused by FGFR1 fusion proteins resulting from a subset of acquired translocations that target chromosome band 8p11. These chimeric proteins have constitutive FGFR1 tyrosine kinase activity and are believed to deregulate hemopoietic development in a manner analogous to BCR-ABL in chronic myeloid leukemia. Here we have studied the role of STAT proteins in transformation mediated by the most common of these fusions, ZNF198-FGFR1. We found that STATs 1, 3, and 5 were activated constitutively in ZNF198-FGFR1-transformed Ba/F3 cells and that STATs 2, 4, and 6 were also tyrosine-phosphorylated. Induction of dominant negative STAT mutants showed that activation of STAT5, but not STATs 1 or 3, was essential for the anti-apoptotic effect of ZNF198-FGFR1 and that STAT5 activation is essential for the elevated levels of BclXL in transformed cells. STAT5 activation was also shown to be required for continued cell cycle progression of BaF3/ZNF198-FGFR1 cells in conditions of cytokine deprivation and for up-regulation of the DNA repair protein Rad51. These findings suggest a critical role of STAT5 activation in transformation mediated by ZNF198-FGFR1.     

ZNF198, a zinc finger protein rearranged in myeloproliferative disease, localizes to the PML nuclear bodies and interacts with SUMO-1 and PML

The ZNF198/FGFR1 fusion gene in atypical myeloproliferative disease produces a constitutively active cytoplasmic tyrosine kinase, unlike ZNF198 which is normally a nuclear protein. We have now shown that the ZNF198/FGFR1 fusion kinase interacts with the endogenous ZNF198 protein suggesting that the function of ZNF198 may be compromised in cells expressing it. Little is currently known about the endogenous function of ZNF198 and to investigate this further we performed a yeast two-hybrid analysis and identified SUMO-1 as a binding partner of ZNF198. These observations were confirmed using co-immunoprecipitation which demonstrated that ZNF198 is covalently modified by SUMO-1. Since many of the SUMO-1-modified proteins are targeted to the PML nuclear bodies we used confocal microscopy to show that SUMO-1, PML and ZNF198 colocalize to punctate structures, shown by immunocytochemistry to be PML bodies. Using co-immunoprecipitation we now show that PML and sumoylated ZNF198 can be found in a protein complex in the cell. Mutation of the SUMO-1 binding site in wild-type ZNF198 resulted in loss of distinct PML bodies, reduced PML levels and a more dispersed nuclear localization of the PML protein. In cells expressing ZNF198/FGFR1, which also lack the SUMO-1 binding site, SUMO-1 is preferentially localized in the cytoplasm, which is associated with loss of distinct PML bodies. Recently, arsenic trioxide (ATO) was proposed as an alternative therapy for APL that was resistant to traditional therapy. Treatment of cells expressing ZNF198/FGFR1 with ATO demonstrated reduced autophosphorylation of the ZNF198/FGFR1 protein and induced apoptosis, which is not seen in cells expressing wild-type ZNF198. Overall our results suggest that the sumoylation of ZNF198 is important for PML body formation and that the abrogation of sumoylation of ZNF198 in ZNF198/FGFR1 expressing cells may be an important mechanism in cellular transformation.     

Negative regulation of hematopoiesis by the fused in myeloproliferative disorders gene product

The t(8;13) translocation, found in a rare and aggressive type of stem cell myeloproliferative disorder, leads to the generation of a fusion protein between the N-terminal gene product of fused in myeloproliferative disorders (FIM)/ZNF198 and the fibroblast growth factor receptor 1 (FGFR1) kinase domain. The chimeric protein was reported to have constitutively activated tyrosine kinase activity. However, little is known about a role of FIM in hematopoietic cell regulation. Here we show that FIM protein is ubiquitously expressed in mouse embryonic tissues but much less in hematopoietic cells. We also show that forced expression of FIM inhibits the emergence of hematopoietic cells in the cultured mouse aorta-gonad-mesonephros (AGM) region on embryonic day (E) 11.5, where definitive hematopoiesis is first found during embryogenesis. These results suggest that the expression level of FIM determines the development of hematopoiesis during mouse ontogeny.     

The Genomic Structure of ZNF198 and Location of Breakpoints in the t(8;13) Myeloproliferative Syndrome

The t(8;13)(p11;q12) is the most common translocation associated with the 8p11 myeloproliferative syndrome and results in an identical mRNA fusion between ZNF198 at 13q12 and FGFR1 at 8p11 in all cases thus far reported. ZNF198 is a widely expressed gene that is predicted to encode a 1377-amino-acid protein with five Zn finger-related motifs known as MYM domains. To determine the genomic DNA structure of ZNF198, we employed bubble PCR from PAC clones with a panel of gene-specific primers. Sequencing of these products revealed that ZNF198 consists of 26 exons with the initiation codon located in exon 4. The t(8;13) results in a consistent mRNA fusion of ZNF198 exon 17 to FGFR1 exon 9. Notable features of the structure of ZNF198 include three noncanonical GC donor splice sites and the presence of an alternatively spliced intron within exon 4. Amplification of genomic DNA from six t(8;13) patients with primers to ZNF198 exon 17 and FGFR1 exon 9 yielded patient-specific products ranging in size from 500 bp to 2.5 kb, indicating that the positions of the breakpoints in the t(8;13) are tightly clustered. The positions of the six t(8;13) breakpoints were determined and found to be distributed across ZNF198 intron 17 and FGFR1 intron 8 with no apparent subclustering. No consistent sequence motifs, repeats, or topoisomerase II cleavage sites were found at or near the breakpoints. It remains unclear why the t(8;13) translocation breakpoints occur within such small genomic regions, and it is possible that strict ZNF198–FGFR1 coding requirements restrict the positions of the breakpoints.     

The oncogenic fusion protein-tyrosine kinase ZNF198/fibroblast growth factor receptor-1 has signaling function comparable with interleukin-6 cytokine receptors

The reciprocal t(8;13) chromosome translocation results in a fusion gene (FUS) in which the N-terminal half of the zinc finger protein ZNF198 is combined with the cytoplasmic domain of the fibroblast growth factor receptor-1 (FGFR1). Expression of FUS is suggested to provide growth-promoting activity to myeloid cells similar to the activity of hematopoietic cytokine receptors. This study determined the specificity of FUS to activate signal transduction pathways. Because no tumor cell line expressing FUS was available, the mode of FUS action was identified in cells transiently and stably transfected with an expression vector for FUS. FUS acted as a constitutively active protein-tyrosine kinase and mediated phosphorylation of STAT1, 3, and 5 but not STAT4 and 6. The same specificity but lower activity was determined for normal FGFR1. STAT activation by FUS, similar to that by interleukin-6-type cytokines, promoted STAT-specific induction of genes. The functionality of FUS, as well as the relative recruitment of STAT isoforms, was determined by the dimerizing function of the zinc finger domain. Replacement of the ZNF198 portion by the Bcr portion as present in the t(8;22) translocation shifted the signaling toward a more prominent STAT5 activation. This study documents that both gene partners forming the fusion oncogene define the activity and the signaling specificity of the protein-tyrosine kinase of FGFR1.     

14‐3‐3 Ligand Prevents Nuclear Import of c‐ABL Protein in Chronic Myeloid Leukemia

Here we demonstrated that the ‘loss of function’ of not‐rearranged c‐ABL in chronic myeloid leukemia (CML) is promoted by its cytoplasmic compartmentalization bound to 14‐3‐3 sigma scaffolding protein. In particular, constitutive tyrosine kinase (TK) activity of p210 BCR‐ABL blocks c‐Jun N‐terminal kinase (JNK) phosphorylation leading to 14‐3‐3 sigma phosphorylation at a critical residue (Ser¹⁸⁶) for c‐ABL binding in response to DNA damage. Moreover, it is associated with 14‐3‐3 sigma over‐expression arising from epigenetic mechanisms (promoter hyper‐acetylation). Accordingly, p210 BCR‐ABL TK inhibition by the TK inhibitor Imatinib mesylate (IM) evokes multiple events, including JNK phosphorylation at Thr¹⁸³, p38 mitogen‐activated protein kinase (MAPK) phosphorylation at Thr¹⁸⁰, c‐ABL de‐phosphorylation at Ser residues involved in 14‐3‐3 binding and reduction of 14‐3‐3 sigma expression, that let c‐ABL release from 14‐3‐3 sigma and nuclear import, and address BCR‐ABL‐expressing cells towards apoptotic death. Informational spectrum method (ISM), a virtual spectroscopy method for analysis of protein interactions based on their structure, and mathematical filtering in cross spectrum (CS) analysis identified 14‐3‐3 sigma/c‐ABL binding sites. Further investigation on CS profiles of c‐ABL‐ and p210 BCR‐ABL‐containing complexes revealed the mechanism likely involved 14‐3‐3 precluded phosphorylation in CML cells.     

Characterization of the SUMO-Binding Activity of the Myeloproliferative and Mental Retardation (MYM)-Type Zinc Fingers in ZNF261 and ZNF198

SUMO-binding proteins interact with SUMO modified proteins to mediate a wide range of functional consequences. Here, we report the identification of a new SUMO-binding protein, ZNF261. Four human proteins including ZNF261, ZNF198, ZNF262, and ZNF258 contain a stretch of tandem zinc fingers called myeloproliferative and mental retardation (MYM)-type zinc fingers. We demonstrated that MYM-type zinc fingers from ZNF261 and ZNF198 are necessary and sufficient for SUMO-binding and that individual MYM-type zinc fingers function as SUMO-interacting motifs (SIMs). Our binding studies revealed that the MYM-type zinc fingers from ZNF261 and ZNF198 interact with the same surface on SUMO-2 recognized by the archetypal consensus SIM. We also present evidence that MYM-type zinc fingers in ZNF261 contain zinc, but that zinc is not required for SUMO-binding. Immunofluorescence microscopy studies using truncated fragments of ZNF198 revealed that MYM-type zinc fingers of ZNF198 are necessary for localization to PML-nuclear bodies (PML-NBs). In summary, our studies have identified and characterized the SUMO-binding activity of the MYM-type zinc fingers in ZNF261 and ZNF198.     

Molecular drug targets in myeloproliferative neoplasms: mutant ABL1, JAK2, MPL, KIT, PDGFRA, PDGFRB and FGFR1

Therapeutically validated oncoproteins in myeloproliferative neoplasms (MPN) include BCR-ABL1 and rearranged PDGFR proteins. The latter are products of intra- ( e.g. FIP1L1-PDGFRA) or inter-chromosomal ( e.g. ETV6-PDGFRB ) gene fusions. BCR-ABL1 is associated with chronic myelogenous leukaemia (CML) and mutant PDGFR with an MPN phenotype characterized by eosinophilia and in addition, in case of FIP1L1-PDGFRA, bone marrow mastocytosis. These genotype-phenotype associations have been effectively exploited in the development of highly accurate diagnostic assays and molecular targeted therapy. It is hoped that the same will happen in other MPN with specific genetic alterations: polycythemia vera ( JAK2 V617F and other JAK2 mutations), essential thrombocythemia ( JAK2 V617F and MPL5 15 mutations), primary myelofibrosis ( JAK2 V617F and MPL515 mutations), systemic mastocytosis ( KIT D816V and other KIT mutations) and stem cell leukaemia/lymphoma ( ZNF198-FGFR1 and other FGFR1 fusion genes). The current review discusses the above-listed mutant molecules in the context of their value as drug targets.     

细胞质内微环境直接影响FGFR1酪氨酸激酶介导的SNT1细胞特异性磷酸化

成纤维细胞生长因子受体（FGFR）介导的SNT1（亦称为FRS2）底物磷酸化具有宿主细胞以及受体特异性。为探明这种宿主细胞特异性的决定因素,我们构建了1个FGFR2IIIb/R1嵌合受体。该嵌合受体具有1个FGFR2IIIb的胞外片段及1个FGFR1蛋白质酪氨酸激酶片段。当表达在3T3细胞（内源性受体为FGFR1并能强烈响应FGFR1）的信号）以及DTE－R1／100细胞时,该嵌合受体能即刻诱导SNT1磷酸化。DTE－R1／100细胞为经长期培养的带有外源性FGFR1的非恶性前列腺肿瘤上皮细胞（DTE）并已获得未转化DTE细胞所不具备的FGFR1信号响应性。与此相反,当表达在非转化DTE细胞或未经长期培养的FGFR1(DTE-R1)锂,FGFR2IIIb/R1嵌合受体则无法诱导SNT1磷酸化。我们曾报导DTE细胞对FGFR1介导的SNT1磷酸化活力及其刺激细胞生长信号的响应性是一种获得性的性质,这种性质的获得与细胞恶化是紧密联系在一起的。在此我们进一步证明FGFR介导的SNT1磷酸化具有宿主细胞特异性。这些结果表明细胞内围绕着激酶的微环境而不是细胞外环境决定了SNT1是否可为FGFR1所磷酸化。而且,长期受外源性FGFR1刺激诱发DTE细胞内微环境的变化,从而使表达在DTE细胞里的FGFR1激酶可强烈地磷酸化SNT1。     

8p12 Stem Cell Myeloproliferative Disorder: the FOP-Fibroblast Growth Factor Receptor 1 Fusion Protein of the t(6;8) Translocation Induces Cell Survival Mediated by Mitogen-Activated Protein Kinase and Phosphatidylinositol 3-Kinase/Akt/mTOR Pathways

The FOP-fibroblast growth factor receptor 1 (FGFR1) fusion protein is expressed as a consequence of a t(6;8) (q27;p12) translocation associated with a stem cell myeloproliferative disorder with lymphoma, myeloid hyperplasia and eosinophilia. In the present report, we show that the fusion of the leucine-rich N-terminal region of FOP to the catalytic domain of FGFR1 results in conversion of murine hematopoietic cell line Ba/F3 to factor-independent cell survival via an antiapoptotic effect. This survival effect is dependent upon the constitutive tyrosine phosphorylation of FOP-FGFR1. Phosphorylation of STAT1 and of STAT3, but not STAT5, is observed in cells expressing FOP-FGFR1. The survival function of FOP-FGFR1 is abrogated by mutation of the phospholipase C gamma binding site. Mitogen-activated protein kinase (MAPK) is also activated in FOP-FGFR1-expressing cells and confers cytokine-independent survival to hematopoietic cells. These results demonstrate that FOP-FGFR1 is capable of protecting cells from apoptosis by using the same effectors as the wild-type FGFR1. Furthermore, we show that FOP-FGFR1 phosphorylates phosphatidylinositol 3 (PI3)-kinase and AKT and that specific inhibitors of PI3-kinase impair its ability to promote cell survival. In addition, FOP-FGFR1-expressing cells show constitutive phosphorylation of the positive regulator of translation p70S6 kinase; this phosphorylation is inhibited by PI3-kinase and mTOR (mammalian target of rapamycin) inhibitors. These results indicate that translation control is important to mediate the cell survival effect induced by FOP-FGFR1. Finally, FOP-FGFR1 protects cells from apoptosis by survival signals including BCL2 overexpression and inactivation of caspase-9 activity. Elucidation of signaling events downstream of FOP-FGFR1 constitutive activation provides insight into the mechanism of leukemogenesis mediated by this oncogenic fusion protein.     

Tyrphostin AG556 increases the activity of large conductance Ca2+‐activated K+ channels by inhibiting epidermal growth factor receptor tyrosine kinase

The present study was designed to investigate whether large conductance Ca²⁺‐activated K⁺ (BK) channels were regulated by epidermal growth factor (EGF) receptor (EGFR) tyrosine kinase. BK current and channel tyrosine phosphorylation level were measured in BK‐HEK 293 cells expressing both functional α‐subunits and the auxiliary β1‐subunits using electrophysiology, immunoprecipitation and Western blotting approaches, respectively, and the function of rat cerebral basilar arteries was determined with a wire myography system. We found that BK current in BK‐HEK 293 cells was increased by the broad spectrum protein tyrosine kinase (PTK) inhibitor genistein and the selective EGFR tyrosine kinase inhibitor AG556, one of the known tyrphostin. The effect of genistein or AG556 was antagonized by the protein tyrosine phosphatase (PTP) inhibitor orthovanadate. On the other hand, orthovanadate or EGF decreased BK current, and the effect was counteracted by AG556. The tyrosine phosphorylation level of BK channels (α‐ and β1‐subunits) was increased by EGF and orthovanadate, while decreased by genistein and AG556, and the reduced tyrosine phosphorylation of BK channels by genistein or AG556 was reversed by orthovanadate. Interestingly, AG556 induced a remarkable enhancement of BK current in rat cerebral artery smooth muscle cells and relaxation of pre‐contracted rat cerebral basilar arteries with denuded endothelium, and these effects were antagonized by the BK channel blocker paxilline or orthovanadate. These results demonstrate that tyrosine phosphorylation of BK channels by EGFR kinase decreases the channel activity, and inhibition of EGFR kinase by AG556 enhances the channel activity and dilates rat cerebral basilar arteries.     

Identification of phosphorylation sites in the COOH‐terminal tail of the μ‐opioid receptor

Phosphorylation is considered a key event in the signalling and regulation of the μ opioid receptor (MOPr). Here, we used mass spectroscopy to determine the phosphorylation status of the C‐terminal tail of the rat MOPr expressed in human embryonic kidney 293 (HEK‐293) cells. Under basal conditions, MOPr is phosphorylated on Ser³⁶³ and Thr³⁷⁰, while in the presence of morphine or [D‐Ala2, NMe‐Phe4, Gly‐ol5]‐enkephalin (DAMGO), the COOH terminus is phosphorylated at three additional residues, Ser³⁵⁶, Thr³⁵⁷ and Ser³⁷⁵. Using N‐terminal glutathione S transferase (GST) fusion proteins of the cytoplasmic, C‐terminal tail of MOPr and point mutations of the same, we show that, in vitro, purified G protein‐coupled receptor kinase 2 (GRK2) phosphorylates Ser³⁷⁵, protein kinase C (PKC) phosphorylates Ser³⁶³, while CaMKII phosphorylates Thr³⁷⁰. Phosphorylation of the GST fusion protein of the C‐terminal tail of MOPr enhanced its ability to bind arrestin‐2 and ‐3. Hence, our study identifies both the basal and agonist‐stimulated phospho‐acceptor sites in the C‐terminal tail of MOPr, and suggests that the receptor is subject to phosphorylation and hence regulation by multiple protein kinases.     

A Phos‐tag SDS‐PAGE method that effectively uses phosphoproteomic data for profiling the phosphorylation dynamics of MEK1

MEK1, an essential component of the mitogen‐activated protein kinase (MAPK) pathway, is phosphorylated during activation of the pathway; 12 phosphorylation sites have been identified in human MEK1 by MS‐based phosphoproteomic methods. By using Phos‐tag SDS‐PAGE, we found that multiple variants of MEK1 with different phosphorylation states are constitutively present in typical human cells. The Phos‐tag‐based strategy, which makes effective use of existing information on the location of phosphorylation sites, permits quantitative time‐course profiling of MEK1 phosphospecies in their respective phosphorylation states. By subsequent immunoblotting with an anti‐HaloTag antibody, we analyzed a HaloTag‐fused MEK1 protein and 12 potential phosphorylation‐site‐directed mutants of the protein transiently expressed in HEK 293 cells. This strategy revealed that MEK1 is constitutively and mainly phosphorylated at the Thr‐292, Ser‐298, Thr‐386, and Thr‐388 residues in vivo, and that combinations of phosphorylations at these four residues produce at least six phosphorylated variants of MEK1. Like the levels of phosphorylation of the Ser‐218 and Ser‐222 residues by RAF1, which have been well studied, the phosphorylation statuses of Thr‐292, Ser‐298, Thr‐386, and Thr‐388 residues vary widely during activation and deactivation of the MAPK pathway. Furthermore, we demonstrated inhibitor‐specific profiling of MEK1 phosphospecies by using three MEK inhibitors: TAK‐733, PD98059, and U0126.     

Identification of the phosphorylation targets of symbiotic receptor‐like kinases using a high‐throughput multiplexed assay for kinase specificity

Detecting the phosphorylation substrates of multiple kinases in a single experiment is a challenge, and new techniques are being developed to overcome this challenge. Here, we used a multiplexed assay for kinase specificity (MAKS) to identify the substrates directly and to map the phosphorylation site(s) of plant symbiotic receptor‐like kinases. The symbiotic receptor‐like kinases nodulation receptor‐like kinase (NORK) and lysin motif domain‐containing receptor‐like kinase 3 (LYK3) are indispensable for the establishment of root nodule symbiosis. Although some interacting proteins have been identified for these symbiotic receptor‐like kinases, very little is known about their phosphorylation substrates. Using this high‐throughput approach, we identified several other potential phosphorylation targets for both these symbiotic receptor‐like kinases. In particular, we also discovered the phosphorylation of LYK3 by NORK itself, which was also confirmed by pairwise kinase assays. Motif analysis of potential targets for these kinases revealed that the acidic motif xxxsDxxx was common to both of them. In summary, this high‐throughput technique catalogs the potential phosphorylation substrates of multiple kinases in a single efficient experiment, the biological characterization of which should provide a better understanding of phosphorylation signaling cascade in symbiosis.     

Quantitative evaluation of His‐tag purification and immunoprecipitation of tristetraprolin and its mutant proteins from transfected human cells

Histidine (His)‐tag is widely used for affinity purification of recombinant proteins, but the yield and purity of expressed proteins are quite different. Little information is available about quantitative evaluation of this procedure. The objective of this study was to evaluate His‐tag procedure quantitatively and to compare it with immunoprecipitation using radiolabeled tristetraprolin (TTP), a zinc finger protein with anti‐inflammatory property. Human embryonic kidney 293 cells were transfected with wild‐type and nine mutant plasmids with single or multiple phosphorylation site mutation(s) in His‐TTP. These proteins were expressed and mainly localized in the cytosol of transfected cells by immunocytochemistry and confocal microscopy. His‐TTP proteins were purified by Ni‐NTA beads with imidazole elution or precipitated by TTP antibodies from transfected cells after being labeled with [³²P]‐orthophosphate. The results showed that (1) His‐tag purification was more effective than immunoprecipitation for TTP purification; (2) mutations in TTP increased the yield of His‐TTP by both purification procedures; and (3) mutations in TTP increased the binding affinity of mutant proteins for Ni‐NTA beads. These findings suggest that bioengineering phosphorylation sites in proteins can increase the production of recombinant proteins. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Interactions of the NPXY microdomains of the low density lipoprotein receptor‐related protein 1

The low density lipoprotein receptor‐related protein 1 (LRP1) mediates internalization of a large number of proteins and protein–lipid complexes and is widely implicated in Alzheimer's disease. The cytoplasmic domain of LRP1 (LRP1‐CT) can be phosphorylated by activated protein‐tyrosine kinases at two NPXY motifs in LRP1‐CT; Tyr 4507 is readily phosphorylated and must be phosphorylated before phosphorylation of Tyr 4473 occurs. Pull‐down experiments from brain lysate revealed numerous proteins binding to LRP1‐CT, but the results were highly variable. To separate which proteins bind to each NPXY motif and their phosphorylation dependence, each NPXY motif microdomain was prepared in both phosphorylated and non‐phosphorylated forms and used to probe rodent brain extracts for binding proteins. Proteins that bound specifically to the microdomains were identified by LC‐MS/MS, and confirmed by Western blot. Recombinant proteins were then tested for binding to each NPXY motif. The NPXY₄₅₀₇ (membrane distal) was found to interact with a large number of proteins, many of which only bound the tyrosine‐phosphorylated form. This microdomain also bound a significant number of other proteins in the unphosphorylated state. Many of the interactions were later confirmed to be direct with recombinant proteins. The NPXY₄₄₇₃ (membrane proximal) bound many fewer proteins and only to the phosphorylated form.