Oncogene (2014) 33, 3463–3472 & 2014 Macmillan Publishers Limited All rights reserved 0950-9232/14 www.nature.com/onc ORIGINAL ARTICLE Coordinate phosphorylation of multiple residues on single AKT1 and AKT2 molecules H Guo1,6, M Gao1,6,YLu1, J Liang1, PL Lorenzi2, S Bai3, DH Hawke4,JLi1, T Dogruluk5, KL Scott5, E Jonasch3, GB Mills1 and Z Ding1 Aberrant AKT activation is prevalent across multiple human cancer lineages providing an important new target for therapy. Twenty- two independent phosphorylation sites have been identified on specific AKT isoforms likely contributing to differential isoform regulation. However, the mechanisms regulating phosphorylation of individual AKT isoform molecules have not been elucidated because of the lack of robust approaches able to assess phosphorylation of multiple sites on a single AKT molecule. Using a nanofluidic proteomic immunoassay (NIA), consisting of isoelectric focusing followed by sensitive chemiluminescence detection, we demonstrate that under basal and ligand-induced conditions that the pattern of phosphorylation events is markedly different between AKT1 and AKT2. Indeed, there are at least 12 AKT1 peaks and at least 5 AKT2 peaks consistent with complex combinations of phosphorylation of different sites on individual AKT molecules. Following insulin stimulation, AKT1 was phosphorylated at Thr308 in the T-loop and Ser473 in the hydrophobic domain. In contrast, AKT2 was only phosphorylated at the equivalent sites (Thr309 and Ser474) at low levels. Further, Thr308 and Ser473 phosphorylation occurred predominantly on the same AKT1 molecules, whereas Thr309 and Ser474 were phosphorylated primarily on different AKT2 molecules. Although basal AKT2 phosphorylation was sensitive to inhibition of phosphatidylinositol 3-kinase (PI3K), basal AKT1 phosphorylation was essentially resistant. PI3K inhibition decreased pThr451 on AKT2 but not pThr450 on AKT1. Thus, NIA technology provides an ability to characterize coordinate phosphorylation of individual AKT molecules providing important information about AKT isoform-specific phosphorylation, which is required for optimal development and implementation of drugs targeting aberrant AKT activation. Oncogene (2014) 33, 3463–3472; doi:10.1038/onc.2013.301; published online 5 August 2013 Keywords: AKT; phosphorylation; NIA INTRODUCTION between its pleckstrin homology (PH) domain and PtdIns(3,4,5)P3, The serine/threonine kinase AKT (also known as protein kinase B, where AKT is phosphorylated at two critical residues, Thr308/309 PKB), comprising a group of three isoforms (AKT1, AKT2 and AKT3), in the activation T-loop and Ser473/474 in the hydrophobic has critical roles in many aspects of cancer pathophysiology domain of AKT1/2 (unless designated otherwise, phosphorylation including cell survival, growth, metabolism and metastasis.1–3 AKT sites are based on the AKT1 amino acid sequence). 3-Phosphoi- is a bona fide oncogene that is frequently activated in cancer nositide-dependent protein kinase 1 (PDK1) (ref. 7) phosphorylates 8 through a variety of mechanisms including amplification of AKT at Thr308, and mTORC2 as well as other potential PDK2 9 growth factor receptors (e.g., HER2/neu, epidermal growth factor phosphorylate AKT at Ser473. Activated AKT then translocates receptor), amplification or mutation of phosphatidylinositol 3- from the cell membrane to other cell compartments to kinase (PI3K), amplification or mutation of AKT isoforms, and phosphorylate its downstream substrates transducing membrane inactivation of phosphatase and tensin homolog or inositol signals to appropriate functional outcomes.10 Phosphorylation of polyphosphate-phosphatase type II (INPP4B).3 Different AKT Thr308 and Ser473 has been proposed to be required for full isoforms appear to mediate critical non-redundant functions in activation of AKT kinase activity.11 However, whether Thr308 and cancer pathophysiology.4–6 For example, AKT1 has been Ser473 phosphorylation is sufficient for full activity or the multiple implicated as a major contributor to tumor initiation, whereas other phosphorylation sites in AKT isoforms are required for AKT2 appears to primarily increase tumor metastasis.5,6 Therefore, processive phosphorylation or modulate the stability, substrate elucidation of the mechanisms regulating AKT activation, especi- access or activity of AKT has not been elucidated.12–14 Further ally AKT isoform-specific activation, will facilitate therapeutic selective phosphorylation of Thr308 and Ser473 alters the approaches to targeting AKT signaling. substrate selectivity of AKT. Thus, an improved understanding of In the canonical AKT activation model, growth factors or other the role of the multiple phosphorylation sites in AKT is required to stimuli activate class I PI3K at the cell membrane to phosphorylate fully elicit the functional regulation of AKT. PtdIns(4,5)P2 to form PtdIns(3,4,5)P3 on the inner cell membrane. In addition to Thr308 and Ser473, currently 20 other residues AKT is then recruited to the cell membrane through interaction of AKT1 have been experimentally validated as sites for 1Department of Systems Biology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA; 2Department of Bioinformatics and Computational Biology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA; 3Department of Genitourinary Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA; 4Department of Molecular Pathology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA and 5Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA. Correspondence: Dr GB Mills or Dr Z Ding, Department of Systems Biology, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030, USA. E-mail: [email protected] or [email protected] 6These authors contributed equally to this work. Received 22 February 2013; revised 4 June 2013; accepted 11 June 2013; published online 5 August 2013 Isoform-specific phosphorylation of AKT H Guo et al 3464 phosphorylation using mass spectrometry or site-specific dominant and one minor peak (Figure 1b). The AKT peaks in approaches, including eight serine residues (122, 124, 126, 129, HCT116 wt could be reconstituted, both in pattern and relative 137, 246, 475 and 477), seven threonine residues (34, 72, 146, 305, magnitude, by combining peaks from AKT2 À / À and AKT1 À / À 312, 450 and 479) and five tyrosine residues (176, 315, 326, 437 cells, validating the use of knockout cells to characterize AKT and 474; http://www.phosphosite.org).12–15 Similarly, AKT2 and isoform-specific phosphorylation. The AKT1 and AKT2 isoform- AKT3 have 22 and 18 validated phosphorylation sites, respectively. specific antibodies also recognized the peaks predicted by the Additional AKT isoform-specific phosphorylation sites may remain studies of HCT116 knockout cells (Supplementary Figures S1A and to be identified. The regulation and importance of phosphoryl- B). The only overlap between the AKT1 and AKT2 protein ation of sites other than Thr308 and Ser473 is only beginning to isoelectronic point (pI) pattern was a peak located at P5.75. be elucidated. For example, phosphorylation at Thr305, Thr312 On the basis of the knockout cell lines and isoform-specific and Tyr474 was shown to contribute to optimal AKT activation. antibody data, it was possible to unambiguously assign the Thr72 and Ser246 have been proposed to be autophosphorylated identity of each pI peak identified with the pan (total) AKT in trans, whereas Thr34, Thr450 and Tyr176 phosphorylation antibody to a specific AKT isoform with the exception of the P5.75 appears to be mediated by upstream kinases including atypical peak, which is present in both AKT1 and AKT2. The ability to assess protein kinase C, c-Jun N-terminal kinases and Ack1.12–14 However, both AKT isoforms with a single antibody greatly aids in the regulation and function of phosphorylation sites other than quantification of relative amounts of each AKT1 or AKT2 peak. Thr308 and Ser473 has not been well studied because of the lack Therefore, unless otherwise indicated, we utilized the pan (total)- of reagents able to assess site-specific phosphorylation. The gap in AKT antibody to establish relative changes in AKT1 and AKT2 knowledge is particularly acute in terms of relative roles of migration. coordinate phosphorylation of sites on individual AKT1, AKT2 and AKT3 molecules. A newly emerging technology, nanofluidic proteomic immuno- Phosphorylation determines AKT pI values assay (NIA), has the potential to characterize protein phosphoryla- As 22 different AKT1 phosphorylation sites have been indepen- tion of multiple different sites and, in particular, coordinate dently identified by either mass spectrometry (19 out of 22; http:// phosphorylation of single AKT molecules including segregating www.phosphosite.org) or alternative approaches (13 out of 22)15 effects on different AKT isoforms.16 NIA combines isoelectric and each of first 10 phosphorylation events will change pI with an focusing of proteins with sensitive chemiluminescence detection expected change of 0.04–0.07 pH units (http://web.expasy.org/ with highly specific antibodies, providing a sensitive and compute_pi/)18 because of the charge added by phosphate, quantitative approach to analyzing protein phosphorylation.16 migration of specific isoforms of AKT on NIA should reflect the The NIA approach demonstrated marked differences in coordinate effects of phosphorylation of single and