Bryan A. Ballif

ATM and ATR Substrate Analysis Reveals Extensive Protein Networks Responsive to DNA Damage

Cellular responses to DNA damage are mediated by a number of protein kinases, including ATM (ataxia telangiectasia mutated) and ATR (ATM and Rad3-related). The outlines of the signal transduction portion of this pathway are known, but little is known about the physiological scope of the DNA damage response (DDR). We performed a large-scale proteomic analysis of proteins phosphorylated in response to DNA damage on consensus sites recognized by ATM and ATR and identified more than 900 regulated phosphorylation sites encompassing over 700 proteins. Functional analysis of a subset of this data set indicated that this list is highly enriched for proteins involved in the DDR. This set of proteins is highly interconnected, and we identified a large number of protein modules and networks not previously linked to the DDR. This database paints a much broader landscape for the DDR than was previously appreciated and opens new avenues of investigation into the responses to DNA damage in mammals.

mTOR and S6K1 Mediate Assembly of the Translation Preinitiation Complex through Dynamic Protein Interchange and Ordered Phosphorylation Events

In response to nutrients, energy sufficiency, hormones, and mitogenic agents, S6K1 phosphorylates several targets linked to translation. However, the molecular mechanisms whereby S6K1 is activated, encounters substrate, and contributes to translation initiation are poorly understood. We show that mTOR and S6K1 maneuver on and off the eukaryotic initiation factor 3 (eIF3) translation initiation complex in a signal-dependent, choreographed fashion. When inactive, S6K1 associates with the eIF3 complex, while the S6K1 activator mTOR/raptor does not. Cell stimulation promotes mTOR/raptor binding to the eIF3 complex and phosphorylation of S6K1 at its hydrophobic motif. Phosphorylation results in S6K1 dissociation, activation, and subsequent phosphorylation of its translational targets, including eIF4B, which is then recruited into the complex in a phosphorylation-dependent manner. Thus, the eIF3 preinitiation complex acts as a scaffold to coordinate a dynamic sequence of events in response to stimuli that promote efficient protein synthesis.

Identification of the FANCI Protein, a Monoubiquitinated FANCD2 Paralog Required for DNA Repair

Fanconi anemia (FA) is a developmental and cancer-predisposition syndrome caused by mutations in genes controlling DNA interstrand crosslink repair. Several FA proteins form a ubiquitin ligase that controls monoubiquitination of the FANCD2 protein in an ATR-dependent manner. Here we describe the FA protein FANCI, identified as an ATM/ATR kinase substrate required for resistance to mitomycin C. FANCI shares sequence similarity with FANCD2, likely evolving from a common ancestral gene. The FANCI protein associates with FANCD2 and, together, as the FANCI-FANCD2 (ID) complex, localize to chromatin in response to DNA damage. Like FANCD2, FANCI is monoubiquitinated and unexpectedly, ubiquitination of each protein is important for the maintenance of ubiquitin on the other, indicating the existence of a dual ubiquitin-locking mechanism required for ID complex function. Mutation in FANCI is responsible for loss of a functional FA pathway in a patient with Fanconi anemia complementation group I.

Abraxas and RAP80 Form a BRCA1 Protein Complex Required for the DNA Damage Response

The BRCT repeats of the breast and ovarian cancer predisposition protein BRCA1 are essential for tumor suppression. Phosphopeptide affinity proteomic analysis identified a protein, Abraxas, that directly binds the BRCA1 BRCT repeats through a phospho-Ser-X-X-Phe motif. Abraxas binds BRCA1 to the mutual exclusion of BACH1 (BRCA1-associated C-terminal helicase) and CtIP (CtBP-interacting protein), forming a third type of BRCA1 complex. Abraxas recruits the ubiquitin-interacting motif (UIM)–containing protein RAP80 to BRCA1. Both Abraxas and RAP80 were required for DNA damage resistance, G2-M checkpoint control, and DNA repair. RAP80 was required for optimal accumulation of BRCA1 on damaged DNA (foci) in response to ionizing radiation, and the UIM domains alone were capable of foci formation. The RAP80-Abraxas complex may help recruit BRCA1 to DNA damage sites in part through recognition of ubiquitinated proteins.

Phosphoproteomic Analysis of the Developing Mouse Brain

Proper development of the mammalian brain requires the precise integration of numerous temporally and spatially regulated stimuli. Many of these signals transduce their cues via the reversible phosphorylation of downstream effector molecules. Neuronal stimuli acting in concert have the potential of generating enormous arrays of regulatory phosphoproteins. Toward the global profiling of phosphoproteins in the developing brain, we report here the use of a mass spectrometry-based methodology permitting the first proteomic-scale phosphorylation site analysis of primary animal tissue, identifying over 500 protein phosphorylation sites in the developing mouse brain.

Rsk1 mediates a MEK–MAP kinase cell survival signal

BACKGROUND Growth factors activate an array of cell survival signaling pathways. Mitogen-activated protein (MAP) kinases transduce signals emanating from their upstream activators MAP kinase kinases (MEKs). The MEK-MAP kinase signaling cassette is a key regulatory pathway promoting cell survival. The downstream effectors of the mammalian MEK-MAP kinase cell survival signal have not been previously described. RESULTS We identify here a pro-survival role for the serine/threonine kinase Rsk1, a downstream target of the MEK-MAP kinase signaling pathway. In cells that are dependent on interleukin-3 (IL-3) for survival, pharmacological inhibition of MEKs antagonized the IL-3 survival signal. In the absence of IL-3, a kinase-dead Rsk1 mutant eliminated the survival effect afforded by activated MEK. Conversely, a novel constitutively active Rsk1 allele restored the MEK-MAP kinase survival signal. Experiments in vitro and in vivo demonstrated that Rsk1 directly phosphorylated the pro-apoptotic protein Bad at the serine residues that, when phosphorylated, abrogate Bads pro-apoptotic function. Constitutively active Rsk1 caused constitutive Bad phosphorylation and protection from Bad-modulated cell death. Kinase-inactive Rsk1 mutants antagonize Bad phosphorylation. Bad mutations that prevented phosphorylation by Rsk1 also inhibited Rsk1-mediated cell survival. CONCLUSIONS These data support a model in which Rsk1 transduces the mammalian MEK-MAP kinase signal in part by phosphorylating Bad.

Fyn tyrosine kinase is a critical regulator of disabled-1 during brain development.

BACKGROUND Disabled-1 (Dab1) is an intracellular adaptor protein that regulates migrations of various classes of neurons during mammalian brain development. Dab1 function depends on its tyrosine phosphorylation, which is stimulated by Reelin, an extracellular signaling molecule. Reelin increases the stoichiometry of Dab1 phosphorylation and downregulates Dab1 protein levels. Reelin binds to various cell surface receptors, including two members of the low-density lipoprotein receptor family that also bind to Dab1. Mutations in Dab1, its phosphorylation sites, Reelin, or the Reelin receptors cause a common phenotype. However, the molecular mechanism whereby Reelin regulates Dab1 tyrosine phosphorylation is poorly understood. RESULTS We found that Reelin-induced Dab1 tyrosine phosphorylation in neuron cultures is inhibited by acute treatment with pharmacological inhibitors of Src family, but not Abl family, kinases. In addition, Reelin stimulates Src family kinases by a mechanism involving Dab1. We analyzed the Dab1 protein level and tyrosine phosphorylation stoichiometry by using brain samples and cultured neurons that were obtained from mouse embryos carrying mutations in Src family tyrosine kinases. We found that fyn is required for proper Dab1 levels and phosphorylation in vivo and in vitro. When fyn copy number is reduced, src, but not yes, becomes important, reflecting a partial redundancy between fyn and src. CONCLUSIONS Reelin activates Fyn to phosphorylate and downregulate Dab1 during brain development. The results were unexpected because Fyn deficiency does not cause the same developmental phenotype as Dab1 or Reelin deficiency. This suggests additional complexity in the Reelin signaling pathway.

The Chlamydomonas reinhardtii BBSome is an IFT cargo required for export of specific signaling proteins from flagella

The Bardet-Biedl syndrome protein complex (BBSome) is a cargo adapter rather than an essential part of the intraflagellar transport (IFT) machinery.

SKAR is a specific target of S6 kinase 1 in cell growth control.

BACKGROUND The mammalian target of rapamycin (mTOR) and phosphatidylinositol 3-kinase (PI3K) signaling pathways promote cell growth and cell cycle progression in response to nutritional, energy, and mitogenic cues. In mammalian cells, the ribosomal protein S6 kinases, S6K1 and S6K2, lie downstream of mTOR and PI3K, suggesting that translational control through the phosphorylation of S6 regulates cell growth. Interestingly, genetic experiments predict that a substrate that is specific to S6K1 but not S6K2 regulates cell growth. RESULTS Here we identify SKAR as a novel and specific binding partner and substrate of S6K1 but not S6K2. We find that serines 383 and 385 of human SKAR are insulin-stimulated and rapamycin-sensitive S6K1 phosphorylation sites. Quantitative mass spectrometry reveals that serine 383/385 phosphorylation is sensitive to RNA interference (RNAi)-mediated S6K1 reduction, but not S6K2 reduction. Furthermore, RNAi-mediated reduction of SKAR decreases cell size. SKAR is nuclear protein with homology to the Aly/REF family of RNA binding proteins, which has been proposed to couple transcription with pre-mRNA splicing and mRNA export. CONCLUSIONS We have identified a novel and specific target of S6K1, SKAR, which regulates cell growth. The homology of SKAR to the Aly/REF family links S6K1 with mRNA biogenesis in the control of cell growth.

Regulation of Protein Tyrosine Kinase Signaling by Substrate Degradation during Brain Development

ABSTRACT Disabled-1 (Dab1) is a cytoplasmic adaptor protein that regulates neuronal migrations during mammalian brain development. Dab1 function in vivo depends on tyrosine phosphorylation, which is stimulated by extracellular Reelin and requires Src family kinases. Reelin signaling also negatively regulates Dab1 protein levels in vivo, and reduced Dab1 levels may be part of the mechanism that regulates neuronal migration. We have made use of mouse embryo cortical neuron cultures in which Reelin induces Dab1 tyrosine phosphorylation and Src family kinase activation. We have found that Dab1 is normally stable, but in response to Reelin it becomes polyubiquitinated and degraded via the proteasome pathway. We have established that tyrosine phosphorylation of Dab1 is required for its degradation. Dab1 molecules lacking phosphotyrosine are not degraded in neurons in which the Dab1 degradation pathway is active. The requirements for Reelin-induced degradation of Dab1 in vitro correctly predict Dab1 protein levels in vivo in different mutant mice. We also provide evidence that Dab1 serine/threonine phosphorylation may be important for Dab1 tyrosine phosphorylation. Our data provide the first evidence for how Reelin down-regulates Dab1 protein expression in vivo. Dab1 degradation may be important for ensuring a transient Reelin response and may play a role in normal brain development.