Effect of cell passage and density on protein kinase G expression and activation in vascular smooth muscle cells

It has been shown that rat aortic smooth muscle cells (AoSMCs) lost PKG-I expression when propagated repetitively or grown at low densities. Conversely, AoSMCs isolated from PKG-I deficient mice are indistinguishable from those isolated from normal mice in morphology and growth characteristics. In this study, human AoSMCs were grown from passage 9 (p9) to passage 15 (p15) and rat AoSMCs were isolated and cultured from p1 through p15. Western blotting and immunofluorescence microscopy showed little difference in PKG-I expression among different passages. Next, rat AoSMCs of p4 were grown and harvested at different cell densities. Western blotting again showed little difference among cells seeded or harvested at different densities. To test the effect of cell passage on PKG-I activation, rat AoSMCs of p4 and p11 were treated with cGMP and analyzed by Western blotting for phosphorylated vasodilator-stimulated phosphoprotein (P-VASP). The results showed that p4 had higher level of PKG-I activation than p11.     

Tyrosine phosphorylation inhibits the interaction of 14-3-3 proteins with the plant plasma membrane H+-ATPase

Interaction of 14-3-3 proteins with their targets depends not only on the phosphorylation status of the target but also on that of 14-3-3 (Fu et al., 2000). In this work we demonstrated that the maize 14-3-3 isoform GF14-6 is a substrate of the tyrosine kinase insulin growth factor receptor 1. By means of site-directed mutants of GF14-6, we identified Tyr-137 as the specific tyrosine residue phosphorylated by the insulin growth factor receptor 1. Phosphorylation of GF14-6 on Tyr-137 lowered its affinity for a peptide mimicking the 14-3-3 binding site of the plant plasma membrane H+-ATPase. Moreover, phosphorylation in planta of 14-3-3 tyrosine residues, resulting from incubation with the tyrosine phosphatase inhibitor, phenylarsine oxide, decreased their association to the H+-ATPase.     

Multiple steps of phosphorylation of activated rhodopsin can account for the reproducibility of vertebrate rod single-photon responses

Single-photon responses (SPRs) in vertebrate rods are considerably less variable than expected if isomerized rhodopsin (R*) inactivated in a single, memoryless step, and no other variability-reducing mechanisms were available. We present a new stochastic model, the core of which is the successive ratcheting down of R* activity, and a concomitant increase in the probability of quenching of R* by arrestin (Arr), with each phosphorylation of R* (Gibson, S.K., J.H. Parkes, and P.A. Liebman. 2000. Biochemistry. 39:5738-5749.). We evaluated the model by means of Monte-Carlo simulations of dim-flash responses, and compared the response statistics derived from them with those obtained from empirical dim-flash data (Whitlock, G.G., and T.D. Lamb. 1999. Neuron. 23:337-351.). The model accounts for four quantitative measures of SPR reproducibility. It also reproduces qualitative features of rod responses obtained with altered nucleotide levels, and thus contradicts the conclusion that such responses imply that phosphorylation cannot dominate R* inactivation (Rieke, F., and D.A. Baylor. 1998a. Biophys. J. 75:1836-1857; Field, G.D., and F. Rieke. 2002. Neuron. 35:733-747.). Moreover, the model is able to reproduce the salient qualitative features of SPRs obtained from mouse rods that had been genetically modified with specific pathways of R* inactivation or Ca2+ feedback disabled. We present a theoretical analysis showing that the variability of the area under the SPR estimates the variability of integrated R* activity, and can provide a valid gauge of the number of R* inactivation steps. We show that there is a heretofore unappreciated tradeoff between variability of SPR amplitude and SPR duration that depends critically on the kinetics of inactivation of R* relative to the net kinetics of the downstream reactions in the cascade. Because of this dependence, neither the variability of SPR amplitude nor duration provides a reliable estimate of the underlying variability of integrated R* activity, and cannot be used to estimate the minimum number of R* inactivation steps. We conclude that multiple phosphorylation-dependent decrements in R* activity (with Arr-quench) can confer the observed reproducibility of rod SPRs; there is no compelling need to invoke a long series of non-phosphorylation dependent state changes in R* (as in Rieke, F., and D.A. Baylor. 1998a. Biophys. J. 75:1836-1857; Field, G.D., and F. Rieke. 2002. Neuron. 35:733-747.). Our analyses, plus data and modeling of others (Rieke, F., and D.A. Baylor. 1998a. Biophys. J. 75:1836-1857; Field, G.D., and F. Rieke. 2002. Neuron. 35:733-747.), also argue strongly against either feedback (including Ca2+-feedback) or depletion of any molecular species downstream to R* as the dominant cause of SPR reproducibility.     

CAMP-dependent protein kinase inhibits proline transport across the rat renal tubular brush border membrane

Very little is known about the cellular mechanisms controlling renal tubular amino acid transport. cAMP-dependent protein kinase (cAK) modulates the activity of several ion channels and pumps in biological membranes. The direct influence of cAK on transmembrane amino acid transport has not been investigated. We studied the effect the cAK-mediated phosphorylation on Na+- and Cl–-linked proline transport across the rat renal brush border membrane (BBM). cAK bioassay and Western hybridization analysis using cAK subunit-specific antibodies demonstrated the presence of the enzyme in the BBM. Brush border membrane vesicles (BBMV) were phosphorylated using the hyposmotic shock technique. cAMP, by activating endogenous cAK,and exogenous, highly purified catalytic subunit of cAK inhibited NaCl-dependent proline transport by phosphorylated, lysed/resealed BBMV compared with control vesicles. The cAK-mediated inhibition of proline uptake was completely abolished when phosphorylation at the cytoplasmic (inner side) of the membrane was prevented by isosmotic, rather than hyposmotic, phosphorylation. The cAK-induced inhibition of proline transport was reversed by the specific cAK inhibitor peptide, PKl. These data suggest that cAMP-dependent protein kinase-mediated phosphorylation modulates Na+- and Cl–-linked proline transport across the tubular luminal membrane.     

Regulation of organelle membrane fusion by Pkc1p

Membrane fusion relies on complex protein machineries, which act in sequence to catalyze the fusion of bilayers. The fusion of endoplasmic reticulum membranes requires the t-SNARE Ufe1p, and the AAA ATPase p97/Cdc48p. While the mechanisms of membrane fusion events have begun to emerge, little is known about how this fusion process is regulated. We provide first evidence that endoplasmic reticulum membrane fusion in yeast is regulated by the action of protein kinase C. Specifically, Pkc1p kinase activity is needed to protect the fusion machinery from ubiquitin-mediated degradation .     

Signalling drought in guard cells

A number of environmental conditions including drought, low humidity, cold and salinity subject plants to osmotic stress. A rapid plant response to such stress conditions is stomatal closure to reduce water loss from plants. From an external stress signal to stomatal closure, many molecular components constitute a signal transduction network that couples the stimulus to the response. Numerous studies have been directed to resolving the framework and molecular details of stress signalling pathways in plants. In guard cells, studies focus on the regulation of ion channels by abscisic acid (ABA), a chemical messenger for osmotic stress. Calcium, protein kinases and phosphatases, and membrane trafficking components have been shown to play a role in ABA signalling process in guard cells. Studies also implicate ABA-independent regulation of ion channels by osmotic stress. In particular, a direct osmosensing pathway for ion channel regulation in guard cells has been identified. These pathways form a complex signalling web that monitors water status in the environment and initiates responses in stomatal movements.     

Prediction of human protein function from post-translational modifications and localization features

We have developed an entirely sequence-based method that identifies and integrates relevant features that can be used to assign proteins of unknown function to functional classes, and enzyme categories for enzymes. We show that strategies for the elucidation of protein function may benefit from a number of functional attributes that are more directly related to the linear sequence of amino acids, and hence easier to predict, than protein structure. These attributes include features associated with post-translational modifications and protein sorting, but also much simpler aspects such as the length, isoelectric point and composition of the polypeptide chain.     

Elusive recognition determinants for ubiquitination

How are proteins recognized as substrates for ubiquitination? Here we summarize insights from recent experiments that address this issue. These highlight the diversity and complexity of determinants for substrate recognition, and raise many questions for further investigation. Copyright © 2002 John Wiley & Sons, Ltd.     

Arg tyrosine kinase is involved in homologous recombinational DNA repair

c-Abl plays important roles in cellular response to DNA damage. However, possible roles for Arg (Abl-related gene) in DNA damage response are unknown. Here, we show that ionizing radiation (IR)-induced Rad51 focus formation is reduced in Arg-deficient cells generated from a chicken B cell line by targeted disruption. This is consistent with the findings that Arg-deficient cells display hypersensitivity to IR, elevated frequencies of IR-induced chromosomal aberrations, and reduced targeted integration frequencies. All of these abnormalities in DNA damage repair are also observed in ATM-deficient cells but not in c-Abl-deficient cells. Finally, we show that Arg interacts with and phosphorylates Rad51 in 293T cells. These results suggest that Arg plays a role in homologous recombinational (HR) DNA repair by phosphorylating Rad51.