Hypo-phosphorylation of the retinoblastoma protein (pRb) by cyclin D:Cdk4/6 complexes results in active pRb

In cycling cells, the retinoblastoma protein (pRb) is un- and/or hypo-phosphorylated in early G1 and becomes hyper-phosphorylated in late G1. The role of hypo-phosphorylation and identity of the relevant kinase(s) remains unknown. We show here that hypo-phosphorylated pRb associates with E2F in vivo and is therefore active. Increasing the intracellular concentration of the Cdk4/6 specific inhibitor p15INK4b by transforming growth factor β treatment of keratinocytes results in G1 arrest and loss of hypo-phosphorylated pRb with an increase in unphosphorylated pRb. Conversely, p15INK4b-independent transforming growth factor β-mediated G1 arrest of hepatocellular carcinoma cells results in loss of Cdk2 kinase activity with continued Cdk6 kinase activity and pRb remains only hypo-phosphorylated. Introduction of the Cdk4/6 inhibitor p16INK4a protein into cells by fusion to a protein transduction domain also prevents pRb hypo-phosphorylation with an increase in unphosphorylated pRb. We conclude that cyclin D:Cdk4/6 complexes hypo-phosphorylate pRb in early G1 allowing continued E2F binding.

A transformation-associated complex involving tyrosine kinase signal adapter proteins and caldesmon links v-ErbB signaling to actin stress fiber disassembly

The avian erythroblastosis viral oncogene (v-erbB) encodes a receptor tyrosine kinase that possesses sarcomagenic and leukemogenic potential. We have expressed transforming and nontransforming mutants of v-erbB in fibroblasts to detect transformation-associated signal transduction events. Coimmunoprecipitation and affinity chromatography have been used to identify a transformation-associated, tyrosine phosphorylated, multiprotein complex. This complex consists of Src homologous collagen protein (Shc), growth factor receptor binding protein 2 (Grb2), son of sevenless (Sos), and a novel tyrosine phosphorylated form of the cytoskeletal regulatory protein caldesmon. Immunofluorescence localization studies further reveal that, in contrast to the distribution of caldesmon along actin stress fibers in normal fibroblasts, caldesmon colocalizes with Shc in plasma membrane blebs in transformed fibroblasts. This colocalization of caldesmon and Shc correlates with actin stress fiber disassembly and v-erbB-mediated transformation. The tyrosine phosphorylation of caldesmon, and its association with the Shc–Grb2–Sos signaling complex directly links tyrosine kinase oncogenic signaling events with cytoskeletal regulatory processes, and may define one mechanism regulating actin stress fiber disassembly in transformed cells.

Down-regulation of interferon γ-activated STAT1 by UV light

STAT1 is a cytoplasmic transcription factor that is phosphorylated by Janus kinases (Jak) in response to interferon-γ (IFNγ). Phosphorylated STAT1 translocates to the nucleus, where it turns on specific sets of IFNγ-inducible genes. Here, we show that UV light interferes with tyrosine phosphorylation of STAT1, thereby hindering IFNγ from exerting its biological effects. This effect is not due to a down-regulation of the IFNγ receptor because phosphorylation of upstream-located Jak1 and Jak2 was not suppressed by UV light. In contrast, UV light had no effect on the phosphorylation of STAT3, which is activated by the proinflammatory cytokine interleukin 6. The UV light effect on STAT1 phosphorylation could be antagonized by vanadate, indicating at least partial involvement of a protein tyrosine phosphatase. Therefore, this study indicates a mechanism by which UV light can inhibit gene activation and suggests STAT1 as a new extranuclear UV target closely located to the membrane.

Role of tyrosine phosphorylation of a cellular protein in adeno-associated virus 2-mediated transgene expression

The adeno-associated virus 2 (AAV), a single-stranded DNA-containing, nonpathogenic human parvovirus, has gained attention as a potentially useful vector for human gene therapy. However, the single-stranded nature of the viral genome significantly impacts upon the transduction efficiency, because the second-strand viral DNA synthesis is the rate-limiting step. We hypothesized that a host-cell protein interacts with the single-stranded D sequence within the inverted terminal repeat structure of the AAV genome and prevents the viral second-strand DNA synthesis. Indeed, a cellular protein has been identified that interacts specifically and preferentially with the D sequence at the 3′ end of the AAV genome. This protein, designated the single-stranded D-sequence-binding protein (ssD-BP), is phosphorylated at tyrosine residues and blocks AAV-mediated transgene expression in infected cells by inhibiting the leading strand viral DNA synthesis. Inhibition of cellular protein tyrosine kinases by genistein results in dephosphorylation of the ssD-BP, leading not only to significant augmentation of transgene expression from recombinant AAV but also to autonomous replication of the wild-type AAV genome. Dephosphorylation of the ssD-BP also correlates with adenovirus infection, or expression of the adenovirus E4orf6 protein, which is known to induce AAV DNA replication and gene expression. Thus, phosphorylation state of the ssD-BP appears to play a crucial role in the life cycle of AAV and may prove to be an important determinant in the successful use of AAV-based vectors in human gene therapy.

Phosphorylation of two regulatory tyrosine residues in the activation of Bruton’s tyrosine kinase via alternative receptors

Mutation of Bruton’s tyrosine kinase (Btk) impairs B cell maturation and function and results in a clinical phenotype of X-linked agammaglobulinemia. Activation of Btk correlates with an increase in the phosphorylation of two regulatory Btk tyrosine residues. Y551 (site 1) within the Src homology type 1 (SH1) domain is transphosphorylated by the Src family tyrosine kinases. Y223 (site 2) is an autophosphorylation site within the Btk SH3 domain. Polyclonal, phosphopeptide-specific antibodies were developed to evaluate the phosphorylation of Btk sites 1 and 2. Crosslinking of the B cell antigen receptor (BCR) or the mast cell Fcɛ receptor, or interleukin 5 receptor stimulation each induced rapid phosphorylation at Btk sites 1 and 2 in a tightly coupled manner. Btk molecules were singly and doubly tyrosine-phosphorylated. Phosphorylated Btk comprised only a small fraction (≤5%) of the total pool of Btk molecules in the BCR-activated B cells. Increased dosage of Lyn in B cells augmented BCR-induced phosphorylation at both sites. Kinetic analysis supports a sequential activation mechanism in which individual Btk molecules undergo serial transphosphorylation (site 1) then autophosphorylation (site 2), followed by successive dephosphorylation of site 1 then site 2. The phosphorylation of conserved tyrosine residues within structurally related Tec family kinases is likely to regulate their activation.

Myelin basic protein kinase activity in tomato leaves is induced systemically by wounding and increases in response to systemin and oligosaccharide elicitors

In response to wounding, a 48-kDa myelin basic protein (MBP) kinase is activated within 2 min, both locally and systemically, in leaves of young tomato plants. The activating signal is able to pass through a steam girdle on the stem, indicating that it moves through the xylem and does not require intact phloem tissue. A 48-kDa MBP kinase is also activated by the 18-amino acid polypeptide systemin, a potent wound signal for the synthesis of systemic wound response proteins (swrps). The kinase activation by systemin is strongly inhibited by a systemin analog having a Thr-17 → Ala-17 substitution, which is a powerful antagonist of systemin activation of swrp genes. A 48-kDa MBP kinase activity also increases in response to polygalacturonic acid and chitosan but not in response to jasmonic acid or phytodienoic acid. In def1, a mutant tomato line having a defective octadecanoid pathway, the 48-kDa MBP kinase is activated by wounding and systemin as in the wild-type plants. This indicates that MBP kinase functions between the perception of primary signals and the DEF1 gene product. In response to wounding, the MBP kinase is phosphorylated on phosphotyrosine residues, indicating a relationship to the mitogen-activated protein kinase family of protein kinases.

High-affinity binding of the Drosophila Numb phosphotyrosine-binding domain to peptides containing a Gly-Pro-(p)Tyr motif

The phosphotyrosine-binding (PTB) domain is a recently identified protein module that has been characterized as binding to phosphopeptides containing an NPXpY motif (X = any amino acid). We describe here a novel peptide sequence recognized by the PTB domain from Drosophila Numb (dNumb), a protein involved in cell fate determination and asymmetric cell division during the development of the Drosophila nervous system. Using a Tyr-oriented peptide library to screen for ligands, the dNumb PTB domain was found to bind selectively to peptides containing a YIGPYφ motif (φ represents a hydrophobic residue). A synthetic peptide containing this sequence bound specifically to the isolated dNumb PTB domain in solution with a dissociation constant (Kd) of 5.78 ± 0.74 μM. Interestingly, the affinity of this peptide for the dNumb PTB domain was increased (Kd = 1.41 ± 0.10 μM) when the second tyrosine in the sequence was phosphorylated. Amino acid substitution studies of the phosphopeptide demonstrated that a core motif of sequence GP(p)Y is required for high-affinity binding to the dNumb PTB domain. Nuclear magnetic resonance experiments performed on isotopically labeled protein complexed with either Tyr- or pTyr-containing peptides suggest that the same set of amino acids in the dNumb PTB domain is involved in binding both phosphorylated and nonphosphorylated forms of the peptide. The in vitro selectivity of the dNumb PTB domain is therefore markedly different from those of the Shc and IRS-1 PTB domains, in that it interacts preferentially with a GP(p)Y motif, rather than NPXpY, and does not absolutely require ligand phosphorylation for binding. Our results suggest that the PTB domain is a versatile protein module, capable of exhibiting varied binding specificities.

A single amino acid substitution converts a carboxylesterase to an organophosphorus hydrolase and confers insecticide resistance on a blowfly

Resistance to organophosphorus (OP) insecticides is associated with decreased carboxylesterase activity in several insect species. It has been proposed that the resistance may be the result of a mutation in a carboxylesterase that simultaneously reduces its carboxylesterase activity and confers an OP hydrolase activity (the “mutant ali-esterase hypothesis”). In the sheep blowfly, Lucilia cuprina, the association is due to a change in a specific esterase isozyme, E3, which, in resistant flies, has a null phenotype on gels stained using standard carboxylesterase substrates. Here we show that an OP-resistant allele of the gene that encodes E3 differs at five amino acid replacement sites from a previously described OP-susceptible allele. Knowledge of the structure of a related enzyme (acetylcholinesterase) suggests that one of these substitutions (Gly137 → Asp) lies within the active site of the enzyme. The occurrence of this substitution is completely correlated with resistance across 15 isogenic strains. In vitro expression of two natural and two synthetic chimeric alleles shows that the Asp137 substitution alone is responsible for both the loss of E3’s carboxylesterase activity and the acquisition of a novel OP hydrolase activity. Modeling of Asp137 in the homologous position in acetylcholinesterase suggests that Asp137 may act as a base to orientate a water molecule in the appropriate position for hydrolysis of the phosphorylated enzyme intermediate.

Cloning of a novel T-cell protein FYB that binds FYN and SH2-domain-containing leukocyte protein 76 and modulates interleukin 2 production

T cell receptor ζ (TcRζ)/CD3 ligation initiates a signaling cascade that involves src kinases p56lck and ζ-associated protein 70, leading to the phosphorylation of substrates such as TcRζ, Vav, SH2-domain-containing leukocyte protein 76 (SLP-76), cbl, and p120/130. FYN binding protein (FYB or p120/130) associates with p59fyn, the TcRζ/CD3 complex, and becomes tyrosine-phosphorylated in response to receptor ligation. In this study, we report the cDNA cloning of human and murine FYB and show that it is restricted in expression to T cells and myeloid cells and possesses an overall unique hydrophilic sequence with several tyrosine-based motifs, proline-based type I and type II SH3 domain binding motifs, several putative lysine/glutamic acid-rich nuclear localization motifs, and a SH3-like domain. In addition to binding the src kinase p59fyn, FYB binds specifically to the hematopoietic signaling protein SLP-76, an interaction mediated by the SLP-76 SH2 domain. In keeping with this, expression of FYB augmented interleukin 2 secretion from a T cell hybridoma, DC27.10, in response to TcRζ/CD3 ligation. FYB is therefore a novel hematopoietic protein that acts as a component of the FYN and SLP-76 signaling cascades in T cells.

Slow cycling of unphosphorylated myosin is inhibited by calponin, thus keeping smooth muscle relaxed

A key unanswered question in smooth muscle biology is whether phosphorylation of the myosin regulatory light chain (RLC) is sufficient for regulation of contraction, or if thin-filament-based regulatory systems also contribute to this process. To address this issue, the endogenous RLC was extracted from single smooth muscle cells and replaced with either a thiophosphorylated RLC or a mutant RLC (T18A/S19A) that cannot be phosphorylated by myosin light chain kinase. The actin-binding protein calponin was also extracted. Following photolysis of caged ATP, cells without calponin that contained a nonphosphorylatable RLC shortened at 30% of the velocity and produced 65% of the isometric force of cells reconstituted with the thiophosphorylated RLC. The contraction of cells reconstituted with nonphosphorylatable RLC was, however, specifically suppressed in cells that contained calponin. These results indicate that calponin is required to maintain cells in a relaxed state, and that in the absence of this inhibition, dephosphorylated cross-bridges can slowly cycle and generate force. These findings thus provide a possible framework for understanding the development of latch contraction, a widely studied but poorly understood feature of smooth muscle.

Postnatal neuronal proliferation in mice lacking Ink4d and Kip1 inhibitors of cyclin-dependent kinases

Development of the central nervous system requires proliferation of neuronal and glial cell precursors followed by their subsequent differentiation in a highly coordinated manner. The timing of neuronal cell cycle exit and differentiation is likely to be regulated in part by inhibitors of cyclin-dependent kinases. Overlapping and sustained patterns of expression of two cyclin-dependent kinases, p19Ink4d and p27Kip1, in postmitotic brain cells suggested that these proteins may be important in actively repressing neuronal proliferation. Animals derived from crosses of Ink4d- null with Kip1-null mice exhibited bradykinesia, proprioceptive abnormalities, and seizures, and died at about 18 days after birth. Metabolic labeling of live animals with bromodeoxyuridine at postnatal days 14 and 18, combined with immunolabeling of neuronal markers, showed that subpopulations of central nervous system neurons were proliferating in all parts of the brain, including normally dormant cells of the hippocampus, cortex, hypothalamus, pons, and brainstem. These cells also expressed phosphorylated histone H3, a marker for late G2 and M-phase progression, indicating that neurons were dividing after they had migrated to their final positions in the brain. Increased proliferation was balanced by cell death, resulting in no gross changes in the cytoarchitecture of the brains of these mice. Therefore, p19Ink4d and p27Kip1 cooperate to maintain differentiated neurons in a quiescent state that is potentially reversible.

Independent regulation of JNK/p38 mitogen-activated protein kinases by metabolic oxidative stress in the liver

The stress-activated protein kinases JNK and p38 mediate increased gene expression and are activated by environmental stresses and proinflammatory cytokines. Using an in vivo model in which oxidative stress is generated in the liver by intracellular metabolism, rapid protein–DNA complex formation on stress-activated AP-1 target genes was observed. Analysis of the induced binding complexes indicates that c-fos, c-jun, and ATF-2 were present, but also two additional jun family members, JunB and JunD. Activation of JNK precedes increased AP-1 DNA binding. Furthermore, JunB was shown to be a substrate for JNK, and phosphorylation requires the N-terminal activation domain. Unexpectedly, p38 activity was found to be constitutively active in the liver and was down-regulated through selective dephosphorylation following oxidative stress. One potential mechanism for p38 dephosphorylation is the rapid stress-induced activation of the phosphatase MKP-1, which has high affinity for phosphorylated p38 as a substrate. These data demonstrate that there are mechanisms for independent regulation of the JNK and p38 mitogen-activated protein kinase signal transduction pathways after metabolic oxidative stress in the liver.

An Eph receptor regulates integrin activity through R-Ras

The ability of integrins to mediate cell attachment to extracellular matrices and to blood proteins is regulated from inside the cell. Increased ligand-binding activity of integrins is critical for platelet aggregation upon blood clotting and for leukocyte extravasation to inflamed tissues. Decreased adhesion is thought to promote tumor cell invasion. R-Ras, a small intracellular GTPase, regulates the binding of integrins to their ligands outside the cell. Here we show that the Eph receptor tyrosine kinase, EphB2, can control integrin activity through R-Ras. Cells in which EphB2 is activated become poorly adherent to substrates coated with integrin ligands, and a tyrosine residue in the R-Ras effector domain is phosphorylated. The R-Ras phosphorylation and loss of cell adhesion are causally related, because forced expression of an R-Ras variant resistant to phosphorylation at the critical site made cells unresponsive to the anti-adhesive effect of EphB2. This is an unusual regulatory pathway among the small GTPases. Reduced adhesiveness induced through the Eph/R-Ras pathway may explain the repulsive effect of the Eph receptors in axonal pathfinding and may facilitate tumor cell invasion and angiogenesis.

Stress-induced phosphorylation of STAT1 at Ser727 requires p38 mitogen-activated protein kinase whereas IFN-γ uses a different signaling pathway

STAT1 is an essential transcription factor for macrophage activation by IFN-γ and requires phosphorylation of the C-terminal Ser727 for transcriptional activity. In macrophages, Ser727 phosphorylation in response to bacterial lipopolysaccharide (LPS), UV irradiation, or TNF-α occurred through a signaling path sensitive to the p38 mitogen-activated protein kinase (p38 MAPK) inhibitor SB203580 whereas IFN-γ-mediated Ser727 phosphorylation was not inhibited by the drug. Consistently, SB203580 did not affect IFN-γ-mediated, Stat1-dependent transcription but inhibited its enhancement by LPS. Furthermore, LPS, UV irradiation, and TNF-α caused activation of p38 MAPK whereas IFN-γ did not. An essential role for p38 MAPK activity in STAT1 Ser727 phosphorylation was confirmed by using cells expressing an SB203580-resistant p38 MAPK. In such cells, STAT1 Ser727 phosphorylation in response to UV irradiation was found to be SB203580 insensitive. Targeted disruption of the mapkap-k2 gene, encoding a kinase downstream of p38 MAPK with a key role in LPS-stimulated TNF-α production and stress-induced heat shock protein 25 phosphorylation, was without a significant effect on UV-mediated Ser727 phosphorylation. The recombinant Stat1 C terminus was phosphorylated in vitro by p38MAPKα and β but not by MAPK-activated protein kinase 2. Janus kinase 2 activity, previously reported to be required for IFN-γ-mediated Ser727 phosphorylation, was not needed for LPS-mediated Ser727 phosphorylation, and activation of Janus kinase 2 did not cause the appearance of STAT1 Ser727 kinase activity. Our data suggest that STAT1 is phosphorylated at Ser727 by a stress-activated signaling pathway either through p38 MAPK directly or through an unidentified kinase downstream of p38MAPK.

Mammalian capping enzyme complements mutant Saccharomyces cerevisiae lacking mRNA guanylyltransferase and selectively binds the elongating form of RNA polymerase II

5′-Capping is an early mRNA modification that has important consequences for downstream events in gene expression. We have isolated mammalian cDNAs encoding capping enzyme. They contain the sequence motifs characteristic of the nucleotidyl transferase superfamily. The predicted mouse and human enzymes consist of 597 amino acids and are 95% identical. Mouse cDNA directed synthesis of a guanylylated 68-kDa polypeptide that also contained RNA 5′-triphosphatase activity and catalyzed formation of RNA 5′-terminal GpppG. A haploid strain of Saccharomyces cerevisiae lacking mRNA guanylyltransferase was complemented for growth by the mouse cDNA. Conversion of Lys-294 in the KXDG-conserved motif eliminated both guanylylation and complementation, identifying it as the active site. The K294A mutant retained RNA 5′-triphosphatase activity, which was eliminated by N-terminal truncation. Full-length capping enzyme and an active C-terminal fragment bound to the elongating form and not to the initiating form of polymerase. The results document functional conservation of eukaryotic mRNA guanylyltransferases from yeast to mammals and indicate that the phosphorylated C-terminal domain of RNA polymerase II couples capping to transcription elongation. These results also explain the selective capping of RNA polymerase II transcripts.