Site-directed mutagenesis was used to remove a critical phosphorylation site, Thr-197, near the active site of the catalytic subunit of cAMP-dependent protein kinase. This residue is present in a number of protein kinases, and its phosphorylation largely influences catalytic activity. We changed Thr-197 to aspartic acid and alanine and measured the effects of these substitutions on the kinetic mechanism and inhibitor affinities. The mutants were expressed as the free catalytic subunit and as soluble fusion proteins of glutathione-S-transferase. The values for KATP and Kpeptide for all three mutants are raised by approximately 2 orders of magnitude relative to the wild-type enzyme. Viscosometric measurements indicate that elevations in Kpeptide are the result of reduced rates for phosphoryl transfer and not reduced substrate affinities. This implies that the loop that contains the phosphothreonine, the activation loop, does not reduce access to the substrate site as proposed for the inactive forms of cdk2 kinase [DeBont, H. L., et al. (1993) Nature 363, 595-602] and MAP kinase [Zhang, F., et al. (1994) Nature 367, 704-711]. The mutants associate slowly with the wild-type regulatory subunit, although the cAMP-free wild-type regulatory subunit inhibits the mutants stoichiometrically. A mutant regulatory subunit that binds cAMP poorly and rapidly inhibits the wild-type catalytic subunit does not inhibit the mutant proteins. These data suggest that the phosphothreonine region serves as a docking surface for the regulatory subunit in the holoenzyme complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Unlike mammals, birds regenerate auditory hair cells (HCs) after injury. During regeneration, mature non-sensory supporting cells (SCs) leave quiescence and convert into HCs, through non-mitotic or mitotic mechanisms. During embryogenesis, Notch ligands from nascent HCs exert lateral inhibition, restricting HC production. Here, we examined whether Notch signalling (1) is needed in mature birds to maintain the HC/SC pattern in the undamaged auditory epithelium or (2) governs SC behavior once HCs are injured. We show that Notch pathway genes are transcribed in the mature undamaged epithelium, and after HC injury, their transcription is upregulated in the region of highest mitotic activity. In vitro treatment with DAPT, an inhibitor of Notch activity, had no effect on SCs in the undamaged epithelium. Following HC damage, DAPT had no direct effect on SC division. However, after damage, DAPT caused excessive regeneration of HCs at the expense of SCs, through both mitotic and non-mitotic mechanisms. Conversely, overexpression of activated Notch in SCs after damage caused them to maintain their phenotype and inhibited HC regeneration. Therefore, signalling through Notch is not required for SC quiescence in the healthy epithelium or to initiate HC regeneration after damage. Rather, Notch prevents SCs from regenerating excessive HCs after damage.

Aminoglycoside (AG) antibiotics are widely used to prevent life-threatening infections, and cisplatin is used in the treatment of various cancers, but both are ototoxic and result in loss of sensory hair cells from the inner ear. ORC-13661 is a new drug that was derived from PROTO-1, a compound first identified as protective in a large-scale screen utilizing hair cells in the lateral line organs of zebrafish larvae. Here, we demonstrate, in zebrafish larvae and in mouse cochlear cultures, that ORC-13661 provides robust protection of hair cells against both ototoxins, the AGs and cisplatin. ORC-13661 also prevents both hearing loss in a dose-dependent manner in rats treated with amikacin and the loading of neomycin-Texas Red into lateral line hair cells. In addition, patch-clamp recordings in mouse cochlear cultures reveal that ORC-13661 is a high-affinity permeant blocker of the mechanoelectrical transducer (MET) channel in outer hair cells, suggesting that it may reduce the toxicity of AGs by directly competing for entry at the level of the MET channel and of cisplatin by a MET-dependent mechanism. ORC-13661 is therefore a promising and versatile protectant that reversibly blocks the hair cell MET channel and operates across multiple species and toxins.

Since a basic surface on the catalytic (C) subunit of cAMP-dependent protein kinase is important for binding to the regulatory (R) subunit, acidic residues in R were sought that might contribute to R-C interaction. Using differential labeling by a water-soluble carbodiimide (Buechler, T. A., and Taylor, S. S. (1990) Biochemistry 29, 1937-1943), seven specific carboxylates in RI␣ were identified that were protected from chemical modification in the holoenzyme; each was then replaced with Ala. Of these, rRI(E15A/E106A/D107A)), rRI(E105A), rRI(D140A), rRI(E143A), and rRI(D258A) all were defective in holoenzyme formation and define negative electrostatic surfaces on RI␣. An additional conserved carboxylate, Glu 101 in RI␣ and the equivalent, Glu 99 in RII␣ were mutated to Ala. Replacement of Glu 101 had no effect while rRII(E99A) was very defective. RI␣ and RII␣ thus differ in the molecular details of how they recognize C. Unlike wild-type RI, two additional mutants, rRI(D170A) and rRI(K242A), inhibited C-subunit stoichiometrically in the presence of cAMP and show increases in both on-and off-rates. Asp 170 , which contributes directly to the hydrogen bonding network in cAMPbinding site A, thus contributes also to holoenzyme stability.Cyclic AMP-dependent protein kinase (cAPK) 1 exists as an inactive tetramer (R 2 C 2 ) that dissociates in the presence of cAMP to release two active catalytic (C) subunits and a dimeric regulatory (R) subunit saturated with four molecules of cAMP (R 2 -cAMP 4 ). The two classes of cAPK, type I and type II, differ primarily in their regulatory subunits (1). The two known families of physiological inhibitors of cAPK, the regulatory subunits (RI and RII) and the heat-stable protein kinase inhibitor (PKI) employ a common mechanism for the inhibition of C. Each inhibitor has an autoinhibitory domain that resembles the substrate consensus sequence, "RRXS/T⌿," where X is any amino acid, and the Pϩ1 site is a hydrophobic group (⌿). In RII the P-site is a Ser, while in RI and PKI, this site is an Ala (2-4).Although binding of this autoinhibitor region to the active site of C is essential for inhibition, this interaction alone is not sufficient for high affinity binding. The R-subunits, as well as PKI, bind to C with K d values that lie in the subnanomolar range (5-7), while peptides containing only the autoinhibitor site bind with micromolar affinity (8, 9). Furthermore, mutant forms of RI and RII with Ala substitutions at the P-2 and P-3 arginines can still form stable holoenzyme complexes in the absence of cAMP (10, 11), indicating that there are additional regions of interaction between R and C that lie beyond the autoinhibitor site. These sites are referred to here as peripheral sites.As shown in Fig. 1, the R-subunit has a well defined domain structure consisting of an N-terminal dimerization domain, followed by the autoinhibitory region and two tandem cAMPbinding domains, A and B. Proteolytic cleavage of the RIIsubunit at a site just prior to the P-3 Arg yielded a monomeric R-subunit fra...

The receptor for leukemia inhibitory factor (LIF) consists of two polypeptides, the LIF receptor and gp130. Agonist stimulation has been shown previously to cause phosphorylation of gp130 on serine, threonine, and tyrosine residues. We found that gp130 fusion proteins were phosphorylated exclusively on Ser-782 by LIF-and growth factor-stimulated 3T3-L1 cell extracts. Ser-780 was required for phosphorylation of Ser-782 but was not itself phosphorylated. Ser-782 is located immediately Nterminal to the di-leucine motif of gp130, which regulates internalization of the receptor. Transient expression of chimeric granulocyte colony-stimulating factor receptor (G-CSFR)-gp130(S782A) receptors resulted in increased cell surface expression in COS-7 cells and increased ability to induce vasoactive intestinal peptide gene expression in IMR-32 neuroblastoma cells when compared with expression of chimeric receptors containing wild-type gp130 cytoplasmic domains. These results identify Ser-782 as the major phosphorylated serine residue in human gp130 and indicate that this site regulates cell surface expression of the receptor polypeptide. Leukemia inhibitory factor (LIF)1 is a multifunctional cytokine that mediates a variety of physiological effects in numerous cell lineages. For example, LIF stimulates leukemic cell differentiation and the proliferation of myeloid and platelet precursors, inhibits the differentiation of embryonic stem cells, stimulates acute phase protein synthesis in hepatocytes, inhibits lipoprotein lipase activity in adipocytes, stimulates myoblast proliferation, and converts sympathetic neurons from noradrenergic to cholinergic phenotypes (Refs. 1-3 and the references therein). LIF, ciliary neurotrophic factor, oncostatin M, interleukin-11, cardiotrophin-1, and interleukin-6 make up a distinct subgroup of the cytokine superfamily that all exhibit a four-anti-parallel ␣-helical bundle structure arranged in a two-up, two-down configuration and associate with the shared signaling molecule, gp130, after initial binding to their unique low affinity ␣-receptor binding subunits (1-4).LIF signals through a heterodimeric receptor complex consisting of LIFR and gp130 (5). Lacking intrinsic enzymatic activity (6), initiation of LIF receptor signaling occurs in a manner analogous to that of activated growth factor receptors (7, 8). LIF-stimulated dimerization of LIFR and gp130 results in activation of the Jak/Tyk family of nonreceptor protein tyrosine kinases (4, 9). The activated Jaks then phosphorylate and activate the signal transducers and activators of transcription (STATs), which regulate a variety of cytokine-responsive genes (10 -13). Activated LIF receptors have also been shown to recruit and stimulate numerous SH2-containing signaling molecules, including phospholipase C-␥, Shc, Grb2, phosphoinositol 3-kinase, pp120, and the protein-tyrosine phosphatase SHP-2 (14).Activated LIF receptors also stimulate several components of the MAP kinase cascade, including MAPK kinase, the MAPK isozymes ERK1 and ERK2, and ...

The catalytic (C) subunit of cAMP-dependent protein kinase requires two distinct surfaces to form a stable complex with its physiological inhibitors, the regulatory (R) subunits and the heat-stable protein kinase inhibitors. In addition to a substrate-like segment that is common to both inhibitors, R requires a peripheral recognition site, PRS2. This surface is comprised of the essential phosphorylation site, Thr-197, His-87, Trp-196, and several surrounding basic residues. To probe the role of Trp-196 in the recognition of R, Trp-196 was replaced with Arg and Ala. Although both rC(W196A) and rC(W196R) were inhibited readily with cAMP-free R, they failed to form an inhibited holoenzyme complex with native R under conditions in which wild-type holoenzyme formed readily. Pairing rC(W196R) with mutant forms of R lacking domain B or having defects in cAMP binding sites A or B highlighted the importance of the conformation of R, and, in particular, the accessibility of site A. One of these mutants, rR(R333K), having a defect in cAMP binding site B formed a stable complex with rC(W196R) in the absence of cAMP. However, unlike wild-type holoenzyme, this complex was active.Cyclic AMP-dependent protein kinase (cAPK) 1 is a tightly regulated enzyme that plays a multitude of roles in growth regulation, gene transcription, and metabolism. Of equal physiological importance to the specificity of this enzyme for its protein substrates is its ability to be turned on and off in response to cellular signals. There are two known physiological inhibitors of cAPK, the regulatory subunits (RI and RII) and the heat-stable protein kinase inhibitors (PKIs) (1). The cooperative binding of cAMP to the inactive tetrameric holoenzyme, (R 2 C 2 ), causes the complex to dissociate, releasing two active catalytic subunits (C) and a dimeric regulatory subunit saturated with four molecules of cAMP (R 2 cAMP 4 ). PKIs interact only with the free C subunit and carry, in addition to a cAPK inhibition site, a nuclear export signal that shuttles the complex actively out of the nucleus (2).The R subunits and PKIs inhibit C by a common mechanism. Both contain an inhibitory consensus sequence, RRXS/T⌿, that is shared by most substrates and inhibitors of cAPK, where the phosphorylation (P) site is either Ser, Thr, or Ala, X is any amino acid, and ⌿, at the P ϩ 1 site, is a hydrophobic residue. In RI and PKI, the P site is an Ala, whereas in RII, this site is a phosphorylatable Ser (3-5). This consensus site sequence binds competitively to the active site cleft and is similar for both R and PKI. However, this interaction is not sufficient to achieve high affinity binding. Binding to C is bipartite. Both R and PKI require an additional interaction site.As indicated in Fig. 1, the C subunit contains two distinct additional surfaces on the large lobe that are important for recognition of PKI and R. One surface, referred to here as peripheral recognition site 1 (PRS1), is essential for high affinity binding of PKI, and another is required for high affinity ...

Two classes of molecules inhibit the catalytic subunit (C) of the cyclic AMP-dependent protein kinase (cAPK), the heat-stable protein kinase inhibitors (PKIs) and the regulatory (R) subunits. Basic sites on C, previously identified as important for R/C interaction in yeast TPKI and corresponding to Lys213, Lys217, and Lys189 in murine Ca, were replaced with either Ala or Thr and characterized for their kinetic properties and ability to interact with RI and PKI. rC(K213A) and rC(K217A) were both defective in forming holoenzyme with RI but were inhibited readily with PKI. This contrasts with rC(R133A), which is defective in binding PKI but not RI (Wen & Taylor, 1994). Thus, the C-subunit employs two distinct electrostatic surfaces to achieve high-affinity binding with these two types of inhibitory molecules even though all inhibitors share a common consensus site that occupies the active site cleft. Unlike TPKI, mutation of Lys189 had no effect. The mutant C subunits that were defective in binding RI, rC(K213A) and rC(K217A), were then paired with three RI mutants, rRI(D140A), rRI(E143A), and rRI(D258A), shown previously to be defective in recognition of C. Although the mutations at Asp140 and Asp258 in RI were additive with respect to the C mutations, rC(K213A) and rRI(E143A) were compensatory, thus identifying a specific electrostatic interaction site between RI and C. The results are discussed in terms of the RI and C crystal structures and the sequence homology between the yeast and mammalian enzymes.

Leukemia inhibitory factor (LIF) and ciliary neurotrophic factor (CNTF) are cytokines which signal through receptor complexes that include the receptor subunits glycoprotein 130 (gp130) and the LIF receptor (LIFR), but CNTF also requires the non-signal transducing CNTF receptor (CNTFR) for binding. We show here that in IMR-32 neuronal cells endogenously expressing the receptor subunits for LIF and CNTF, CNTFR, but not gp130 or LIFR, is found in detergent-resistant lipid rafts. In addition, stimulation of these cells with CNTF resulted in a rapid translocation of a portion of gp130 and LIFR into detergent-resistant lipid rafts while an equivalent stimulation with LIF did not. Disruption of lipid rafts by cholesterol depletion of cell membranes blocked the CNTF-induced translocation of LIFR and gp130. Interestingly, while cholesterol-depletion did not inhibit signal transducer and activator of transcription 3 phosphorylation by either CNTF or LIF stimulation, it strongly inhibited both CNTF-and LIF-mediated phosphorylation of extracellular signal-regulated kinases 1 and 2 and Akt. LIF and CNTF generally appear to have redundant effects in cells responsive to both cytokines. Intriguingly, the data presented here suggest a possible mechanism whereby CNTF or other cytokines that signal through CNTFR could generate signals distinct from those elicited by cytokines such as LIF which utilize a LIFR/gp130 heterodimer, via association with or exclusion from lipid rafts.