We have examined the interactions of the p85 regulatory subunit of phosphatidylinositol 3-kinase with the endothelium-specific Flt-1 receptor tyrosine kinase using the yeast two-hybrid system. We find that both the amino- and carboxyl-terminal SH2 domains of p85 bind to Flt-1. We have performed site-directed mutagenesis on the carboxyl-terminal tail of the Flt-1 receptor in order to identify the site(s) that is responsible for the p85 interactions. A single tyrosine to phenylalanine change at position 1213 inhibits the binding of both p85 SH2 domains. Phosphopeptide mapping of the wild type and mutant protein expressed in insect cells verifies that this amino acid is a target for autophosphorylation. The amino acids following this tyrosine are VNA and thus define a novel binding site for p85.

Reoviruses induce apoptosis both in cultured cells and in vivo. Apoptosis plays a major role in the pathogenesis of reovirus encephalitis and myocarditis in infected mice. Reovirus-induced apoptosis is dependent on the activation of transcription factor NF-B and downstream cellular genes. To better understand the mechanism of NF-B activation by reovirus, NF-B signaling intermediates under reovirus control were investigated at the level of Rel, IB, and IB kinase (IKK) proteins. We found that reovirus infection leads initially to nuclear translocation of p50 and RelA, followed by delayed mobilization of c-Rel and p52. This Mammalian reoviruses are nonenveloped viruses that contain a genome of 10 segments of double-stranded RNA (52). Following infection of newborn mice, reovirus disseminates systemically, causing injury to the central nervous system (CNS), heart, and liver (76). Apoptosis induced by reovirus appears to be the primary mechanism for virus-induced encephalitis (53, 54, 59) and myocarditis (22,23,54). Disassembly of internalized virus in the endocytic pathway provides the initial viral trigger for stimulating the signaling pathways that elicit an apoptotic response (19,21).Transcription factor NF-B plays an important regulatory role in apoptosis evoked by reovirus in cultured cells (20) and in vivo (54). Inducible members of the NF-B family are sequestered in the cytoplasm by inhibitory IB proteins, including IB␣, IB␤, IBε, and p100/NF-B2 (3,31,68,79,82). In response to a wide variety of NF-B inducers, IB proteins are phosphorylated at specific serine residues, earmarking these molecules for destruction by the ubiquitin-proteasome pathway (7,12,31,56,75,82). Phosphorylation of IB proteins is mediated by cytokine-inducible IB kinases (IKKs) IKK␣ and IKK␤ (47,78), which can form higher-order complexes containing a regulatory subunit called IKK␥/Nemo (26,50,62,88,91). A primary function of IKK␤ is to modulate the inhibitory interaction of IB␣ with the prototypical form of NF-B containing p50/RelA dimers (25,26,50,58,69). This regulatory circuit, termed the classical pathway of NF-B activation, is strictly dependent on the presence of IKK␥/Nemo (64,65,88). In contrast, IKK␣ functions in an alternative IKK␥-independent pathway of NF-B activation that leads to the proteolytic processing of p100 and the production of a functional p52 Rel subunit (16,66,71). Unlike the classical IKK␤-directed pathway of NF-B activation, the alternative pathway involving IKK␣ is dependent on its prior phosphorylation by NF-Binducing kinase (NIK) (43,66,85). In addition to the cytoplasmic function of IKK␣, a nuclear role for IKK␣ in the transcriptional activation of NF-B-responsive genes has been suggested by in vitro studies (1,32,33,41,48,69,87).To better understand the mechanism of NF-B activation by reovirus, we conducted experiments to define the NF-B/Rel, IB, and IKK proteins that are under reovirus control. These studies revealed that NF-B/Rel proteins are mobilized to the nuclear compartment with biphasic kinetics follo...

Initiation of the genetic programs for inflammation and immunity involves nuclear mobilization of transcription factor NF-B. This signal-dependent process is controlled in part by the ␤-catalytic subunit of IB kinase (IKK␤), which marks IB␣ and other cytoplasmic inhibitors of NF-B for proteolytic destruction. The catalytic activity of IKK␤ is stimulated by pathologic and physiologic inducers of NF-B, such as the Tax oncoprotein and proinflammatory cytokines. We now report evidence that these NF-B inducers target IKK␤ for conjugation to ubiquitin (Ub) in mammalian cells. The apparent molecular size of modified IKK␤ is compatible with monoubiquitination rather than attachment of a multimeric Ub chain. The modification is contingent upon signal-induced phosphorylation of the activation T loop in IKK␤ at Ser-177/Ser-181. The formation of IKK␤-Ub conjugates is disrupted in cells expressing YopJ, a Ub-like protein protease that interferes with the NF-B signaling pathway. These findings indicate an important mechanistic link between phosphorylation, ubiquitination, and the biologic action of IKK␤.The inducible transcription factor NF-B is biochemically coupled to cell-surface members of the tumor necrosis factor (TNF) 1 receptor, Toll-like receptor, and immunoglobulin superfamilies (1). NF-B is persistently activated in cells expressing the Tax protein of human T-cell leukemia virus type 1, which has potent oncogenic properties (2). This deregulated pattern of NF-B activity also underlies acute and chronic inflammatory diseases (1). Nuclear translocation of NF-B is controlled by an inducible multicomponent protein kinase, termed IKK, which targets IB␣ and other cytoplasmic inhibitors of NF-B for proteolytic destruction (3). IKK contains two catalytic subunits, designated IKK␣ and IKK␤, as well as a regulatory subunit called IKK␥ (3). IKK␤ and IKK␥ are essential for proteolytic inactivation of IB␣, whereas IKK␣ mediates NF-B subunit processing via an IKK␥-independent mechanism (4).The catalytic activity of IKK␤ is stimulated by signals that trigger its phosphorylation at Ser-177/Ser-181, such as TNF-␣ (5). These phosphoacceptors lie in a region of the catalytic domain that shares homology with regulatory "T loop" sequences found in mitogen-activated protein kinases and their upstream activators (5). In contrast to its transient pattern of phosphorylation and activation in TNF-stimulated cells, IKK␤ is chronically phosphorylated and activated in cells expressing the Tax oncoprotein (6). Tax-induced activation of IKK␤ is blocked by YopJ (6), a cysteine protease that removes ubiquitin (Ub)-related modifiers from target proteins in mammalian cells (7). This finding with YopJ raised the possibility that IKK␤ might be subject to signal-dependent ubiquitination. In keeping with this possibility, previous studies with partially purified kinase complexes suggested that IKK is conjugated to Ub in vitro (8). However, these pioneering in vitro experiments were conducted prior to molecular cloning of individual IKK subunits (3).We n...

Transcription factor NF-B governs the expression of multiple genes involved in cell growth, immunity, and inflammation. Nuclear translocation of NF-B is regulated from the cytoplasm by IB kinase-␤ (IKK␤), which earmarks inhibitors of NF-B for polyubiquination and proteasome-mediated degradation. Activation of IKK␤ is contingent upon signal-induced phosphorylation of its T loop at Ser-177/Ser-181. T loop phosphorylation also renders IKK␤ a substrate for monoubiquitination in cells exposed to chronic activating cues, such as the Tax oncoprotein or sustained signaling through proinflammatory cytokine receptors. Here we provide evidence that the T loop-proximal residue Lys-163 in IKK␤ serves as a major site for signal-induced monoubiquitination with significant regulatory potential. Conservative replacement of Lys-163 with Arg yielded a monoubiquitination-defective mutant of IKK␤ that retains kinase activity in Tax-expressing cells but is impaired for activation mediated by chronic signaling from the type 1 receptor for tumor necrosis factor-␣. Phosphopeptide mapping experiments revealed that the Lys-163 3 Arg mutation also interferes with proper in vivo but not in vitro phosphorylation of cytokine-responsive serine residues located in the distal C-terminal region of IKK␤. Taken together, these data indicate that chronic phosphorylation of IKK␤ at Ser-177/Ser-181 leads to monoubiquitin attachment at nearby Lys-163, which in turn modulates the phosphorylation status of IKK␤ at select C-terminal serines. This mechanism for post-translational cross-talk may play an important role in the control of IKK␤ signaling during chronic inflammation.

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