Haploinsufficiency of the hematopoietic transcription factor GATA2 underlies monocytopenia and mycobacterial infections; dendritic cell, monocyte, B, and natural killer (NK) lymphoid deficiency; familial myelodysplastic syndromes (MDS)/acute myeloid leukemia (AML); and Emberger syndrome (primary lymphedema with MDS). A comprehensive examination of the clinical features of GATA2 deficiency is currently lacking. We reviewed the medical records of 57 patients with GATA2 deficiency evaluated at the National Institutes of Health from January 1, 1992, to March 1, 2013, and categorized mutations as missense, null, or regulatory to identify genotype-phenotype associations. We identified a broad spectrum of disease: hematologic (MDS 84%, AML 14%, chronic myelomonocytic leukemia 8%), infectious (severe viral 70%, disseminated mycobacterial 53%, and invasive fungal infections 16%), pulmonary (diffusion 79% and ventilatory defects 63%, pulmonary alveolar proteinosis 18%, pulmonary arterial hypertension 9%), dermatologic (warts 53%, panniculitis 30%), neoplastic (human papillomavirus+ tumors 35%, Epstein-Barr virus+ tumors 4%), vascular/lymphatic (venous thrombosis 25%, lymphedema 11%), sensorineural hearing loss 76%, miscarriage 33%, and hypothyroidism 14%. Viral infections and lymphedema were more common in individuals with null mutations (P = .038 and P = .006, respectively). Monocytopenia, B, NK, and CD4 lymphocytopenia correlated with the presence of disease (P < .001). GATA2 deficiency unites susceptibility to MDS/AML, immunodeficiency, pulmonary disease, and vascular/lymphatic dysfunction. Early genetic diagnosis is critical to direct clinical management, preventive care, and family screening.

The target of rapamycin (TOR), as part of the rapamycinsensitive TOR complex 1 (TORC1), regulates various aspects of protein synthesis. Whether TOR functions in this process as part of TORC2 remains to be elucidated. Here, we demonstrate that mTOR, SIN1 and rictor, components of mammalian (m)TORC2, are required for phosphorylation of Akt and conventional protein kinase C (PKC) at the turn motif (TM) site. This TORC2 function is growth factor independent and conserved from yeast to mammals. TM site phosphorylation facilitates carboxyl-terminal folding and stabilizes newly synthesized Akt and PKC by interacting with conserved basic residues in the kinase domain. Without TM site phosphorylation, Akt becomes protected by the molecular chaperone Hsp90 from ubiquitination-mediated proteasome degradation. Finally, we demonstrate that mTORC2 independently controls the Akt TM and HM sites in vivo and can directly phosphorylate both sites in vitro. Our studies uncover a novel function of the TOR pathway in regulating protein folding and stability, processes that are most likely linked to the functions of TOR in protein synthesis.

Summary Protein kinase C (PKC) is a family of kinases that plays diverse roles in many cellular functions, notably proliferation, differentiation, and cell survival. PKC is processed by phosphorylation and regulated by cofactor binding and subcellular localization. Extensive detail is available on the molecular mechanisms that regulate the maturation, activation, and signaling of PKC. However, less information is available on how signaling is terminated both from a global perspective and isozyme-specific differences. To target PKC therapeutically, various ATP-competitive inhibitors have been developed, but this method has problems with specificity. One possible new approach to developing novel, specific therapeutics for PKC would be to target the signaling termination pathways of the enzyme. This review focuses on the new developments in understanding how PKC signaling is terminated and how current drug therapies as well as information obtained from the recent elucidation of various PKC structures and down-regulation pathways could be used to develop novel and specific therapeutics for PKC.

Summary A-Kinase Anchoring Proteins (AKAPs) coordinate cell-signaling events. AKAP79 brings together different combinations of enzyme binding partners to customize the regulation of effector proteins. In neurons muscarinic agonists mobilize an AKAP79-anchored pool of PKC that phosphorylates the KCNQ2 subunit of the M channel. This inhibits potassium permeability to enhance neuronal excitability. Using a dual fluorescent imaging/patch-clamp technique, we visualized AKAP79-anchored PKC phosphorylation of the kinase activity reporter CKAR concurrently with electrophysiological changes in KCNQ2 channels to show that AKAP79 synchronizes both signaling events to optimize the attenuation of M currents. AKAP79 also protects PKC from certain ATP competitive inhibitors. Related studies suggest that context dependent protein-protein interactions alter the susceptibility of another protein kinase, PDK1, to ATP analog inhibitors. This implies that intracellular binding partners not only couple individual molecular events in a cell signaling process but can also change the pharmacological profile of certain protein kinases.

The life cycle of protein kinase C (PKC) is tightly controlled by mechanisms that mature the enzyme, sustain the activationcompetent enzyme, and degrade the enzyme. Here we show that a conserved PXXP motif (Kannan, N., Haste, N., Taylor, S. S., and Neuwald, A. F. (2007) Proc. Natl. Acad. Sci. U. S. A. 104, 1272-1277), in the C-terminal tail of AGC (c-AMP-dependent protein kinase/protein kinase G/protein kinase C) kinases, controls the processing phosphorylation of conventional and novel PKC isozymes, a required step in the maturation of the enzyme into a signaling-competent species. Mutation of both Pro-616 and Pro-619 to Ala in the conventional PKC ␤II abolishes the phosphorylation and activity of the kinase. Co-immunoprecipitation studies reveal that conventional and novel, but not atypical, PKC isozymes bind the chaperones Hsp90 and Cdc37 through a PXXP-dependent mechanism. Inhibitors of Hsp90 and Cdc37 significantly reduce the rate of processing phosphorylation of PKC. Of the two C-terminal sites processed by phosphorylation, the hydrophobic motif, but not the turn motif, is regulated by Hsp90. Overlay of purified Hsp90 onto a peptide array containing peptides covering the catalytic domain of PKC ␤II identified regions surrounding the PXXP segment, but not the PXXP motif itself, as major binding determinants for Hsp90. These Hsp90-binding regions, however, are tethered to the C-terminal tail via a "molecular clamp" formed between the PXXP motif and a conserved Tyr (Tyr-446) in the ␣E-helix. Disruption of the clamp by mutation of the Tyr to Ala recapitulates the phosphorylation defect of mutating the PXXP motif. These data are consistent with a model in which a molecular clamp created by the PXXP motif in the C-terminal tail and determinants in the ␣E-helix of the catalytic domain allows the chaperones Hsp90 and Cdc37 to bind newly synthesized PKC, a required event in the processing of PKC by phosphorylation.Protein kinases, which comprise ϳ2% of the human genome, are key signal transducers that regulate a wide variety of cellular processes, such as growth, proliferation, and metabolism, through catalysis of specific phosphorylation events (1). By integrating signals from extracellular stimuli and transmitting them to targeted downstream substrates, protein kinases serve as a pivotal point of regulation within the cell. Deregulation and mutation of protein kinases play a causal role in human pathology, notably cancer, poising kinases as important targets for the design of therapeutics (2-5). Therefore, understanding the mechanisms that regulate protein kinases, such as those important for maturation and processing, would be critical for designing therapeutics that would maintain the correct functioning of signal transduction pathways.Heat shock proteins (Hsp), 3 such as Hsp90, are ubiquitously expressed molecular chaperones that facilitate protein folding, regulate quality control, and guide protein turnover in an effort to maintain cellular homeostasis (6 -8). Unlike other chaperones such as Hsp70, which non...