Phosphorylation of AKT is altered in GCBCs

We previously showed that signals regulating FOXO1 and the metabolic checkpoint kinase mTORC1 are rewired in GCBCs compared to NBCs¹⁶. Since both FOXO1 and mTORC1 are regulated by AKT, we hypothesized that AKT signaling in GCBCs would be reprogramed to alter these downstream pathways. We previously found that AKT S473 was poorly phosphorylated in both unstimulated NBCs and GCBCs¹⁶. Here we found that, in cells isolated directlyex vivo, total AKT protein amounts were elevated in GCBCs and that AKT T308 was naturally phosphorylated to a greater extent in GCBCs than in NBCs (Fig. 1a,b andSupplementary Fig. 1a,b). PDK1 is the upstream kinase for AKT T308 phosphorylation, and phosphorylation of PDK1 at S241 is essential for its kinase activity²². We found that both total and S241 phosphorylated PDK1 were more abundant in GCBCs compared to NBCs (Fig. 1c). As expected from our prior report¹⁶, AKT S473 phosphorylation was attenuated and transient in GCBCs upon BCR stimulation (Fig. 1d). In contrast, BCR signaling induced robust AKT T308 phosphorylation in GCBCs, comparable to that seen in NBCs (Fig. 1d), despite the fact that BCR signaling in general is markedly attenuated in the GCBCs^15,16 (Fig. 1e). These findings suggest that elevated PDK1 expression and activity in GCBCs might compensate for an attenuated upstream signal to enable efficient AKT p-T308 generation.

Higher PTEN restrains AKT S473 phosphorylation in GCBCs

We then asked why AKT S473 phosphorylation in GCBCs is dampened. AKT S473 phosphorylation is catalyzed by mTORC2, which has been shown to have a markedly lower affinity for PtdIns(3,4,5)P3 compared to PDK1^23,24,25,26. We hypothesized that PtdIns(3,4,5)P₃ generation in GCBCs is controlled to allow efficient activation of PDK1 but not mTORC2. PtdIns(3,4,5)P₃ generation requires PI3K, and in B cells the catalytic subunit p110δ is the major isoform mediating antigen-dependent BCR signaling²⁷. We found that GCBCs expressed more, not less, p110δ protein than NBCs (Supplementary Fig. 2), suggesting the rewired signaling in GCBCs is not due to reduced expression of the catalytic subunit of PI3K. In contrast, PTEN, which negatively regulates AKT activation by converting PtdIns(3,4,5)P₃ to PtdIns(4,5)P₂²⁸, was more highly expressed in GCBCs than in NBCs (Fig. 2a). The finding of elevated PTEN protein in GCBCs suggested that the balance between PtdIns(4,5)P₂ and PtdIns(3,4,5)P₃ might differ between NBCs and GCBCs. To test this, generation of PtdIns(4,5)P₂ and PtdIns(3,4,5)P₃ was measured as a function of time after BCR ligation. Compared to NBC, GCBCs generated less PtdIns(3,4,5)P₃, which returned to baseline within 5 min or less, yet GCBCs accumulated more PtdIns(4,5)P₂ (Fig. 2b), in keeping with higher PTEN amounts in GCBCs. The kinetics of PtdIns(3,4,5)P₃ generation upon BCR ligation correlated very well with attenuated BCR signaling and AKT S473 phosphorylation (Fig. 1d,e andFig. 2b), further suggesting that high PTEN expression in GCBCs might restrain AKT S473 phosphorylation by inhibiting PtdIns(3,4,5)P₃ generation and favoring generation of PtdIns(4,5)P₂. To test this, we used the PTEN inhibitor SF1670. Acute inhibition of PTEN significantly increased PtdIns(3,4,5)P₃ and PtdIns(3,4,)P₂ generation in GCBCs upon BCR stimulation, while, it resulted in a significant decrease in the PTEN-dependent product of PtdIns(3,4,5)P₃ hydrolysis, PtdIns(4,5)P₂ (Fig. 2c). These results were commensurate with our previous finding that SHIP-1 activity is elevated in GCBCs¹⁵; SHIP-1 could limit the accumulation of PtdIns(3,4,5)P₃ by converting PtdIns(3,4,5)P₃ into PtdIns(3,4,)P₂. More importantly, pretreatment with SF1670 increased BCR signal-induced AKT S473 phosphorylation in GCBCs (Fig. 2d), which is dependent on PtdIns(3,4,5)P₃ stimulation of mTORC2 activity. PTEN inhibition also increased p-S6 in GCBCs (Fig. 2d). These findings indicate that suppression of the AKT-mTORC1-S6 axis is mechanistically linked to high PTEN activity in GCBCs.

AKT targets different pathways in GCBCs compared to NBCs

Previous work demonstrated that differential phosphorylation regulates AKT substrate specificity^29,30,31. Since GCBCs and NBC differ in AKT p-T308 and p-S473, we asked whether AKT substrates differ between NBCs and GCBCs. Immunoblotting using an AKT phospho-substrate antibody that specifically recognizes the phosphorylated AKT substrate motif (RXRXXS*/T*) showed that the substrates of AKT in GCBCs qualitatively differed from NBCs as well as activated non-GCBCs (Fig. 3a andSupplementary Fig. 1b).

To obtain a deeper understanding of the GCBC-specific AKT signaling network, we used beads coated with AKT phospho-substrate antibody to immunoprecipitate AKT-phosphorylated substrates from lysates derived from fresh or BCR-stimulated NBCs and GCBCs, followed by a quantitative mass spectrometric assay (Supplementary Fig. 3 andSupplementary Table 1). We identified 33 unique AKT substrates in BCR-activated NBCs and 63 in activated GCBCs, with other patterns of substrate sharing depicted in the Venn diagram (Fig. 3b). In these cell type-specific AKT substrates, we found that AKT preferentially phosphorylated proteins involved in regulating the actin cytoskeleton in NBCs (Fig. 3c). Interestingly, in GCBCs AKT preferentially phosphorylated proteins with functions related to cell control, including transcriptional regulation, RNA processing and signal transduction (Fig. 3c). Together, these results demonstrate that the AKT kinase signaling network is rewired in GCBCs compared to NBCs.

AKT targets proximal BCR signaling regulators in GCBCs

Given that BCR signaling is dampened in GCBCs^15,16,32,33, it was interesting that GCBC-specific AKT targets included negative regulators of BCR signaling, most notably CSK, SHP-1 and HPK1 (Fig. 4a). To evaluate the expression of these three proteins, along with other relevant proteins in the BCR/PI3K signaling cascade, we performed immunoblot analysis with cell lysates of NBCs and GCBCs isolated directlyex vivo (Supplementary Fig. 1b andSupplementary Fig.2). We found that total protein amounts of CSK and HPK1 were higher in GCBCs than NBCs (Supplementary Fig. 2). CSK is the major kinase for the inhibitory tyrosine of LYN, which when phosphorylated maintains LYN in an inactive conformation³⁴. SHP-1 is a phosphatase for multiple activating P-Tyr modifications, including on SYK and BLNK³⁵. HPK1 is a kinase for BLNK, the phosphorylation of which by HPK1 inhibits its linker activity for BCR signal transduction³⁶. The proteomics screen also identified that ARP2, a subunit in the ARP2/3 complex that regulates actin polymerization³⁷, was phosphorylated by AKT in activated NBCs but not GCBCs (Fig. 4a).

To confirm that these proteins were AKT substrates, NBCs and GCBCs were BCR-stimulated with or without an AKT inhibitor, and immunoblotting was used to detect proteins that were immunoprecipitated by the AKT phospho-substrate antibody (Fig. 4b). In agreement with the mass spectrometry results, CSK, SHP-1 and HPK1 were immunoprecipitated by this antibody only in GCBCs, validating these proteins as GCBCs-specific AKT substrates (Fig. 4b). Likely due the different nature of the assays, AKT-dependent phosphorylation of SHP-1 and HPK1 was only found in stimulated GCBCs by immunoblotting, whereas the mass spectrometric screening detected it in stimulated as well as unstimulated GCBCs. Immunoblotting detected ARP2 as an AKT substrate in NBCs only (Fig. 4b), again consistent with the mass spectrometry assay. These results were validated by AKT inhibition, which blocked immunoprecipitation of all these proteins (Fig. 4b right lanes). We included LYN as a negative control, which was not identified by mass spectrometry screening. As expected LYN was not immunoprecipitated by the AKT phospho-substrate antibody under any of the conditions tested (Fig. 4b).

To further confirm results from the mass spectrometry assay, we performedin vitro kinase assays with recombinant activated AKT and found that AKT could effectively phosphorylate recombinant CSK, SHP-1 and HPK1 when ATP was present (Fig. 4c). As a negative control, recombinant AKT did not phosphorylate recombinant LYNin vitro. Together, our mass spectrometry screening and validation experiments suggest that AKT specifically phosphorylates at least three negative regulators of BCR signaling in GCBCs but not NBCs.

AKT phosphorylation enhances negative regulator activity

Next we used mass spectrometry to identify potential AKT phosphorylation sites on CSK, SHP-1 and HPK1, followed by functional assays to determine the consequences of phosphorylation. CSK was phosphorylated by AKT at S284 (Fig. 5a). Phosphorylation of S284 was modeled onto the CSK-c-SRC crystal structure³⁸, which suggested that phosphorylation would enhance the binding between CSK and its substrate and thus might increase CSK kinase activity on LYN and other Src family kinases. We found that recombinant CSK that had been phosphorylated by AKT indeed had a seven-fold higher rate for phosphorylating LYN at Y507 than unphosphorylated CSK (Fig. 5b). AKT phosphorylated SHP-1 on T394, which is within the catalytic domain (Fig. 5c), suggesting functional impact. In fact, enzymatic activity of AKT-phosphorylated SHP-1 was increased by five-fold compared to unmodified SHP-1 (Fig. 5d). This is also consistent with reported increased SHP-1 tyrosine phosphatase activity in GCBCs¹⁵. HPK1 phosphorylates BLNK on T152, which down-modulates BCR signaling³⁶. However, the effect of phosphorylation of HPK1 by AKT has not been documented. We found that AKT-catalyzed phosphorylation sharply increased the activity of HPK1 for phosphorylating BLNK (Fig. 5e). Hence, in each case, AKT-dependent phosphorylation increased the enzymatic activity of negative regulators of BCR signaling.

PTEN inhibition alters AKT signaling networks in GCBCs

To investigate whether elevated PTEN expression in GCBCs is essential for maintaining the GCBC-specific AKT signaling network, we pretreated GCBCs or NBCs with PTEN inhibitor (SF1670) or DMSO before stimulation. We then immunoprecipitated AKT phospho-substrates from cell lysates and performed quantitative mass spectrometric analysis (Supplementary Fig. 3 andSupplementary Table 2). A PANTHER pathway analysis demonstrated that PTEN inhibition substantially altered AKT signaling networks in GCBCs, with ontogenies of signaling, transcription and RNA processing manifesting major effects (Fig. 6a andSupplementary Fig. 4). Specifically, PTEN inhibition reduced the level of AKT phosphorylation of CSK, SHP1 and HPK1 in GCBCs (Fig. 6a,b). On the other hand, AKT-catalyzed ARP2 phosphorylation was increased by PTEN inhibition in GCBCs (Fig. 6a,b). These data link GCBC-specific alterations observed in AKT substrates to PTEN-dependent regulation of phosphatidylinositol phosphate species generation.

AKT inhibition enhances proximal BCR signaling in GCBCs

The proteomic andin vitro kinase assays presented above indicated that AKT in GCBCs could enhance at least three important negative regulators of BCR signaling. We therefore hypothesized that AKT activity in GCBCs negatively controls upstream BCR signaling. The hypothesis predicts that an AKT specific inhibitor would enhance the response to BCR stimulation specifically in GCBCs. Remarkably, AKT inhibition significantly enhanced the amount of active LYN in GCBCs, as measured by the ratio of activating-to-inhibitory tyrosine phosphorylation (Y396/Y507)^34,39 (Fig. 7a). The overall phosphorylation of SYK is a reflection of both LYN activity and SHP-1-mediated phosphatase activity. Our results and model predicted that AKT inhibition would lead to reduced activities of CSK and SHP-1, in turn leading to more LYN kinase activity on SYK as well as less phosphatase activity to hydrolyze p-SYK. Consistent with this, AKT inhibition resulted in notably higher and more persistent amounts of p-SYK in BCR-stimulated GCBCs (Fig. 7a). To indirectly assess the functional effects of AKT inhibition on HPK1, and the resulting effects on generation of signals downstream of BLNK, we measured the generation of both p-BTK and p-PLCγ2. Both were increased by AKT inhibition at all time points after one minute of stimulation (Fig. 7b). Importantly, this effect was GCBC-specific since AKT inhibition did not significantly change the kinetics of p-BTK or p-PLCγ2 in NBCs (Supplementary Fig. 5). These data thus reveal a novel mechanism by which AKT signaling in GCBCs is rewired to form a negative feedback loop acting on proximal BCR signaling and further demonstrate how this operates at multiple levels of the signaling pathway.

Mice and Immunization

B1–8i^+/− BALB/c (referred to as B1–8i), B1–8i^+/−Jκ^−/− BALB/c and IgM B1–8i BCR transgenic BALB/c mice (referred to as MEG) were previously described^15,58,59. Mice of either strain were used as sources of GCBCs and NBCs, as indicated in figure legends. Mice were maintained under specific-pathogen-free conditions supervised by the University of Pittsburgh Institutional Animal Care and Use Committee (IACUC). 6-to-12 weeks old, age- and sex-matched mice were immunized i.p. with 50 μg NP-CGG precipitated in Alum. Mice were analyzed at day 13 to 14 post immunization, or as otherwise indicated in the figure legends.

Cell Preparation and Treatment

NBCs were purified from unimmunized mice and GCBCs were purified from day 13 to 14 NP-CGG immunized mice. The cell purification process was performed as previously described¹⁶. Briefly, splenic NBCs were purified by negative selection using a biotin-conjugated antibody cocktail (antibodies against CD43, CD4, CD8, CD11b, CD11c, Gr-1 and CD138), followed by magnetic bead-depletion of labeled cells (B cell purity ≥ 95%). For GCBC purification from B1–8i mice, biotin-conjugated anti-IgD and anti-CD38 antibodies were added to the B cell purification cocktail; for GCBC purification from MEG mice, biotin-conjugated anti-CD38 antibody was added to the B cell purification cocktail (GCBC purity ≥ 90%). Purified cells or total splenocytes were warmed to 37 °C with 5% CO₂ in B cell medium (RPMI-1640 medium supplemented with 10% FBS, penicillin/streptomycin, glutamine and 50 μM β-mercaptoethanol) for 30 to 40 min and stimulated with 20 μg/ml goat anti-mouse IgM (μ-chain specific, Jackson ImmunoResearch) or 200 ng/ml NP-Ficoll (LGC Biosearch Technologies) as indicated in the figure legends. Endotoxin was removed from antibodies used for stimulation with ToxinEraserTM Endotoxin Removal Kit (GenScript) and endotoxin levels were tested with a LAL assay kit (GenScript) (endotoxin < 0.5 EU/ml). For experiments using inhibitors, cells were treated with 10 μM AKT1/2 kinase inhibitor (Calbiochem) or 10 μM PTEN inhibitor (SF1670, Calbiochem) dissolved in DMSO or the same amount of DMSO alone, as described in figure legends, prior to stimulation. For experiments measuring basal amounts of protein expression or phosphorylation, purified cells were analyzed immediately after purification.

For AKT phospho-substrate antibody immunoblotting, the different cell populations were sorted by FACSAria (BD Immunocytometry Systems). The gating strategy of sorting and post-sort testing is shown inSupplementary Fig. 1b. Briefly, NBCs were identified as CD19⁺CD93^– live singlets;in vivo andin vitro activated B cells as CD19⁺ CD86⁺ live singlets with increased forward scatter; GCBCs were defined as CD19⁺CD95⁺CD38^– live singlets. Purification led in general to over 98% purity of target cell populations. To generatein vitro activated B cells, magnetic bead-purified NBCs (as described above) were cultured in RPMI 1640 media in the presence of 5 μg/ml CpG ODN 1826 (Invivogen) for 48 h at 37 °C. To generatein vivo activated B cells, B1–8i^+/−Jκ^−/− mice were given 50 μg NP-Ficoll (LGC Biosearch Technologies) i.p., and cells were prepared 48 h later.

Immunoblot Analysis

Whole cell lysates were prepared by direct lysing and boiling samples in Laemmli buffer supplemented with β-mercaptoethanol. For the experiment inSupplementary Fig. 2, the same numbers of GCBCs and NBCs were lysed in RIPA buffer supplemented with protease and phosphatase inhibitor cocktail (ThermoFisher Scientific). Protein concentrations were measured with a BCA Protein Assay Kit (Pierce™) and equal amount of protein were loaded in SDS-PAGE gels for immunoblot. The samples were then blotted with the following antibodies against: Actin (clone: C4/actin, BD Biosciences), total-ERK1/2 (clone: 137F5, Cell Signaling Technology), phospho-SYK (Y352; rabbit polyclonal, Cell Signaling Technology), phospho-AKT (T308; clone: D25E6, Cell Signaling Technology), phospho-AKT (S473; clone: D9E, Cell Signaling Technology ), phospho-PDK1 (S241; rabbit polyclonal, Cell Signaling Technology), total-PDK1 (clone: D37A7, Cell Signaling Technology), PTEN (clone: D4.3, Cell Signaling Technology), phospho-LYN (Y396; clone: EP503Y, Abcam), phospho-LYN (Y507; rabbit polyclonal, Cell Signaling Technology), AKT phospho-substrate motif (clone: 110B7E, Cell Signaling Technology), PI(3) Kinase p110δ (clone: D1Q7R, Cell Signaling Technology), PI(3) Kinase p110α (clone: C73F8, Cell Signaling Technology), S6 Ribosomal Protein (clone: 5G10, Cell Signaling Technology), CSK (clone: C74C1, Cell Signaling Technology), CD45 (clone: D4H7K, Cell Signaling Technology), Phospho-Src Family (Tyr416; clone: D49G4, Cell Signaling Technology), Phospho-Src (Tyr527; Rabbit polyclonal, Cell Signaling Technology), total Src (Rabbit polyclonal, Cell Signaling Technology), total SYK (clone: D3Z1E, Cell Signaling Technology), AKT (pan) (clone; C67E7, Cell Signaling Technology), HPK1 (Rabbit polyclonal, Cell Signaling Technology), LYN (clone: C13F9, Cell Signaling Technology), SHP-1 (clone: C14H6, Cell Signaling Technology), ARP2 (clone:D85D5, Cell Signaling Technology) and Phospho-Threonine (clone:D85D5, Cell Signaling Technology). Densitometry was performed using ImageJ software⁶⁰. Immunoblots inSupplementary Fig. 2 were loaded with an equal amount of protein per lane. Actin was used as a normalization loading control for other immunoblots.

Flow Cytometry

Cells were fixed and permeabilized in saponin-based Perm/Wash buffer (Cat#: 554732, BD Biosciences) supplemented with 1.5% paraformaldehyde (PFA) for 10 min at 20–25 °C followed by 30 min on ice. For experiments focused on detecting basal level of phosphorylation, cells were fixed by directly disrupting spleens into RPMI containing 1.5% PFA and incubating for 15 min at 20–25 °C (“instant fix”) before permeabilization with Perm/Wash buffer. Fc receptors on cells were blocked by incubating with anti-CD16/CD32 antibody (clone: 2.4G2, prepared in the lab) prior to staining with fluorochrome-conjugated antibodies against: phospho-S6 (S235/236; clone: D57.2.2E, Cell Signaling Technology), phospho-AKT (S473; clone: M89–61, BD Biosciences), phospho-Btk (Y223/Itk pY180; clone: N35–86, BD Biosciences), phospho-PLC-γ2 (Y759; clone: K86–689.37, BD Biosciences) CXCR4 (clone: L276F12, BioLegend), CD86 (clone: GL1, prepared and conjugated in our lab), B220 (clone: RA3–6B2, BD Bioscience and BioLegend), PNA (Vector Lab, conjugated in our lab), CD95 (clone: Jo2, BD Biosciences), IgM (goat polyclonal, Jackson ImmunoResearch), Igλ (polyclonal, Southern Biotech, conjugated in our lab). For phospho-AKT (T308), cells were stained with unconjugated rabbit anti-mouse phospho-AKT (T308; clone: D25E6, Cell Signaling Technology), followed by secondary Cy3- or Alexa647-conjugated anti-rabbit secondary antibody (ThermoFisher Scientific).

Stained cells were analyzed on a BD LSRII or Fortessa flow cytometer. Data were analyzed with FlowJo 10 software. The following gating strategy was used for cells from B1–8i or MEG mice: GCBCs were gated as B220⁺λ⁺PNA⁺ (or CD95⁺) cells, non-GCBCs were gated as B220⁺λ⁺PNA^– (or CD95^–) cells and NBCs were gated as B220⁺λ^– PNA^– (or CD95^–) cells (Supplementary Fig. 1a).

Measurement of phosphatidylinositol abundance

1 × 10⁶ B cells were collected per time point. Samples were processed per the manufacturers’ instructions included in the Echelon Biosciences Mass ELISA kits for PtdIns(4,5)P₂ (K-4500), PtdIns(3,4,5)P₃ (K-2500s) and PtdIns(3,4)P₂ (K-3800). The mass ELISA assays were measured at 450 nM on a Molecular Devices SpectraMax i3 plate reader. The standard curve was fit assuming a sigmoidal dose-response with variable slope and the amount of phosphatidylinositol in each sample was extrapolated in the GraphPad Prism 7 software package.

Immunoprecipitation of AKT substrates and Mass Spectrometric Analysis

2 × 10⁶ B cells were collected and lysed with a buffer containing 25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP40, 5% glycerol that was supplemented with Roche Complete C protease and PhosStop phosphatase inhibitors. Lysates were sonicated and centrifuged to remove insoluble debris and the remaining lysate was incubated with magnetic bead-conjugated AKT phospho-substrate antibody (Clone: 110B7E, Cell signaling Technologies, Cat #: 8050) for 12 h at 4 °C. The beads were washed twice with lysis buffer and proteins were eluted from the beads with 8 M urea (Sigma, U5128) and 0.1 M Tris-HCl at pH 8.5. The filter aided sample preparation method (FASP) was used to generate tryptic peptides and desalted using C18 spin columns⁶¹. Peptides were suspended in 0.1% formic acid and resolved with liquid chromatography tandem mass spectrometry (LC-MS/MS) using a system comprised of a Waters nanoAcuity HPLC in-line with either a LTQ/OrbitrapVelos Elite hybrid mass spectrometer (Thermo-Fisher) or a Q Exactive mass spectrometer (Thermo-Fisher). Solvent A (0.1% formic acid in HPLC-grade water) and solvent B (0.1% formic acid in 100% acetonitrile) were used as the mobile phase. Peptides were then eluted onto a capillary column (75 μm inner diameter × 360 μm outer diameter × 15 cm long; Polymicro Technologies) 5 μm particle size, 125 pore size C-18 silica-bonded stationary phase (Phenomenex) and resolved using a 100 min gradient at the flow rate of 0.2 μl/min (3–33% B for 90 min, 33–80% B for 2 min, constant at 80% B for 6 min, and then 80–0% B for 2 min). Data were collected in positive ionization mode. PEAKS8 software was used to sequence and identify peptides in each sample using a decoy search at a 1% false discovery rate using the UniProt murine database. Label free quantitation was performed using the quantitative module in the PEAKS8 software.

Bioinformatics

The following criteria was used to identify AKT substrates enriched in different cell types: 1) peptide(s) corresponding to a protein had to be identified in a sample type in the MS analysis and 2) if a given substrate was identified in multiple sample groups, its abundance had to be more than three-fold greater based on the label free quantitation of the mass spectrometric data to be assigned as specific to a group(s). The PANTHER pathway analysis software was used to perform a statistical overrepresentation test to identify biological pathways that were differentially targeted by AKT in the NBCs and GCBCs⁶².

AKT co-immunoprecipitation Analysis

NBCs or GCBCs were activated with anti-IgM antibody in the presence or absence of 10 μM AKT1/2 kinase inhibitor (Calbiochem). Cells were lysed in a buffer containing 25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP40, 5% glycerol that was supplemented with Roche Complete C protease and Roche PhosStop phosphatase inhibitors and incubated with agarose beads coated with magnetic bead-conjugated AKT phospho-substrate antibody (Clone: 110B7E, Cell signaling Technologies, Cat #: 8050) for 2 h at 4 °C. Immunoblot was then used to determine if specific proteins were immunoprecipitated by the antibody. The following antibodies used in immunoblot were all purchased from Cell Signaling Technology: antibodies against CSK (clone: C74C1), SHP-1 (clone: C14H6), HPK1 (rabbit polyclonal), ARP2 (clone: D85D5) and LYN (rabbit polyclonal).

In vitro AKT Enzyme Reactions

Recombinant preactivated AKT (1 μg, Sigma SRP5001) was reacted with 1 mM ATP (Sigma) and 1 μg of recombinant CSK (abcam), SHP-1 (abcam), LYN (abcam) or HPK1 (abcam) for 30 min at 37 °C in an AKT reaction buffer containing 25 mM MOPS, pH 7.2, 12.5 mM glycerol 2-phosphate, 25 mM MgCl₂, 5 mM EGTA,2 mM EDTA and 0.25 mM DTT. Reactions were quenched with Laemmli buffer and resolved by SDS-PAGE. immunoblot was performed using the AKT phospho-substrate antibody to detect AKT mediated phosphorylation of the target proteins. To determine the phosphorylation status of the purchased recombinant pre-activated AKT, we performed an immunoprecipitation with anti-AKT p-S473 followed by immunoblot of the immunoprecipitated and non-immunoprecipitated fractions, along with the input material, using both anti-AKT p-S473 and anti-AKT p-T308, which revealed that the material was a mixture of single and doubly phosphorylated enzyme (data not shown).

Mass spectrometry was used to identify the AKT phosphorylation sites on the substrate as follows:In vitro kinase reactions were performed as detailed above and quenched with a buffer containing 8 M urea and 0.1M Tris-HCl at pH 8.5. FASP was used to prepare tryptic peptides that were analyzed on a LTQ/Orbitrap Velos Elite hybrid mass spectrometer. PEAKS8 software was used to sequence and identify peptides in each sample using a decoy search at a 1% false discovery rate and phospho-serine and phospho-threonine were set as variable modifications.

Enzyme Assays

CSK enzyme assays: 1 μg of recombinant CSK (abcam) was incubated with 1 mM of ATP in the presence or absence (as negative control) of 1 μg of AKT (Sigma) in AKT reaction buffer at 37 °C for 30 min. After dialysis to remove excess ATP and ADP, 0.1 μg equivalent of CSK from the AKT kinase reaction or the negative control reaction was reacted with 1 μg of recombinant LYN (abcam) and 1 mM ATP. Immunoblotting was performed to detect phospho-LYN (Y507) (rabbit polyclonal, Cell Signaling Technology) and total LYN (clone: C13F9, Cell Signaling Technology).

SHP-1 enzyme assays: 1 μg of recombinant SHP-1 (abcam) was incubated with 1 mM of ATP in the presence or absence (as negative control) of 1 μg of AKT (Sigma) in AKT reaction buffer at 37 °C for 30 min. The reactions were dialyzed to remove unreacted ATP and ADP. 0.1 μg of SHP-1 obtained from the reaction with AKT or the negative control reaction was reacted with 50 mM of p-nitrophenyl phosphate (PNPP) in 25 mM HEPES, pH 7.2, 50 mM NaCl, 2.5 mM EDTA, 5 mM DTT. The absorbance at 405 nm was monitored on a Molecular Devices SpectraMax i3 microplate reader.

HPK1 enzyme assays: 1 μg of recombinant HPK1 (abcam) was incubated with 1 mM of ATP in the presence or absence (as negative control) of 1 μg of AKT (Sigma) in AKT reaction buffer at 37 °C for 30 min. After dialysis to remove excess ATP and ADP, the HPK1 reactions were incubated with 1 μg recombinant BLNK. Immunoblot using an antibody that recognized phospho-theronine residues was used to monitor BLNK phosphorylation by HPK1 (rabbit polyclonal, Cell Signaling Technology) and total BLNK protein (clone: D3P2H, Cell Signaling Technology).

Quantification and Statistical Analysis

Statistical analysis was performed with Prism software (GraphPad Software). For comparing two groups,P-values were determined using Student’st-tests (two-tailed). For comparing more than two groups, One-Way ANOVA followed by Tukey test was applied. Two-Way ANOVA followed by Sidak’s multiple comparisons test was used for evaluating data with two factors. Differences between groups were considered significant forP-values < 0.05.

Data availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request. Reagents and methods used in this paper are described in the Life Sciences Reporting Summary, available online.

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Supplementary Materials

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request. Reagents and methods used in this paper are described in the Life Sciences Reporting Summary, available online.