Excitatory interaction between glutamate receptors and protein kinases

One of the most active areas of neurobiology research concerns mechanisms involved in paradigms of synaptic plasticity. A popular model for cellular leaning and memory is long term potentiation (LTP) in hippocamus. LTP requires postsynaptic influx of Ca²⁺ which triggers multiple biochemical pathways resulting in pre- and postsynaptic mechanisms enhancing long term synaptic efficiency. This article focuses on an acute postsynaptic Mechanism that can enhance responsiveness of glutamate receptors. Evidence is presented that calcium/calmodulin/dependent protein kinase II, the major potsynaptic density protein at excitatory glutaminergic synapses, can phosphorylate glutamate receptors and enhance ion current flowing through them. 1994 John Wiley & Sons, Inc.     

Biochemical characterization of the boar sperm 42 kilodalton protein tyrosine kinase: Its potential for tyrosine as well as serine phosphorylation towards microtubule-associated protein 2 and histone H 2B

The majority of cellular responses to changing environmental conditions is regulated by protein kinases. Spermatozoa have many special properties, including motility with demonstrated chemotaxis, the ability to undergo capacitation, and the acrosome reaction, which are in part controlled by extracellular signals and in which sperm kinases are considered to be involved. We have previously reported that there is a protein kinase activity, which phosphorylates the synthetic substrate poly-(Glu, Tyr) with a Km value of 2.3 μM, and is inhibited by the tyrosine kinase inhibitor tyrphostin, in the protein extract from boar spermatozoa (Berruti and Porzio, 1992: Biochim Biophys Acta 1118:149–154). Now we have demonstrated that the enzyme is cytosolic, is active as a monomer of M_r 42,000, is stimulated by Mg²⁺ > Mn²⁺ but not by Ca²⁺, is renaturable, and can phosphorylate native protein substrates such as microtubule-associated protein 2 (MAP2) and histone H2B both on the tyrosine and serine residues. N-terminal sequence analysis suggests that it is a novel protein. These new findings imply that the boar sperm 42 kD kinase may be a novel member of the emerging class of dual-specificity protein kinases, and they raise the intriguing question of its function in the protein kinase network mediating signal transduction in mammalian spermatozoa. © 1994 Wiley-Liss, Inc.     

Differential localization of two isoforms of the regulatory subunit RIIα of cAMP-dependent protein kinase in human sperm: Biochemical and cytochemical study

In the present study, immunogold labeling of ultrathin sections of ejaculated sperm was used to obtain insight into the ultrastructural localization and presumable function of type II cAMP-dependent protein kinase in sperm motion. In the flagellum, a human-specific isoform of the RIIα subunit was located on the axonemal microtubule wall, whereas a different isoform of broader specificity was present in the cytoplasm at the periphery of the coarse fibers and fibrous sheath. This isoform was also found in the mitochondria. The human-specific RIIα subunit is likely linked to microtubules by a unique binding protein of M_r 72kD. These findings are in agreement with the concept of a concerted mechanism involving phosphorylation of both the axonemal microtubules and the fibrous structures for the regulation of mammalian sperm motion. © 1994 Wiley-Liss, Inc.     

Differential accumulation of the transcripts of 22 novel protein kinase genes in Arabidopsis thaliana

22 novel members of the Arabidopsis thaliana protein kinase family (AKs) were identified by using degenerate oligonucleotide primers directed to highly conserved amino acid sequences of the protein kinase (PK) catalytic domain. Of these 22 genes, 16 turned out to carry intron sequences. Homologies of AK sequences were detected to S-locus receptor protein kinases (SRKs) from Brassica spp., to SRK-like PKs from maize and A. thaliana and to several other receptor PKs from A. thaliana. Sequence similarity was also detected to Ca²⁺-dependent PKs (CDPKs) from rape and soybean, to SNF1 and to CDC2 homologues. The genomic organization and the accumulation of the mRNAs from these 22 AK genes were investigated.     

Peptide recognition by PTB and PDZ domains

Protein tyrosine binding (PTB) and ‘post synaptic density disc-large zo-1’ (PDZ) domains bind to short peptidic ligands by augmentation of one of the domain's β sheets and other recognition mechanisms. The two domain classes have a superficial resemblance to each other, even though no sequential homology exists. The structural bases of the interactions are well understood for the domains now experimentally determined, and ligand—target pairs can probably be identified in favorable cases by analogy with the known domains. For both PTB and PDZ classes, functional activities are still not fully defined: it is possible that these domain classes, along with pleckstrin homology domains, have multiple roles.     

HIV-1 Nef protein: purification, crystallizations, and preliminary X-ray diffraction studies.

Human immunodeficiency virus Nef protein accelerates virulent progression of AIDS by its interaction with specific cellular proteins involved in cellular activation and signal transduction. Here we report the purification and crystallization of the conserved core of HIV-1LAI Nef protein in the unliganded form and in complex with the wild-type SH3 domain of the P59fyn protein-tyrosine kinase. One-dimensional NMR experiments show that full-length protein and truncated fragment corresponding to the product of HIV-1 protease cleavage have a well-folded compact tertiary structure. The ligand-free HIV-1 Nefcore protein forms cubic crystals belonging to space group P23 with unit cell dimensions of a = b = c = 86.4 A. The Nef-Fyn SH3 cocrystals belong to the space group P6(1)22 or its enantiomorph, P6(5)22, with unit cell dimensions of a = b = 108.2 A and c = 223.7 A. Both crystal forms diffract to a resolution limit of 3.0 A resolution using synchrotron radiation, and are thus suitable for X-ray structure determination.     

Inhibition of tyrosine phosphorylation potentiates substrate-induced neurite growth

Protein tyrosine kinases (PTKs) have major roles in signal transduction and growth control. There are several lines of evidence implicating PTKs in the regulation of axon growth, and this has led to the suggestion that they are centrally involved in the transduction of neuronal growth signals. To test this idea, we assayed the effect of the compounds genistein and lavendustin, specific inhibitors of PTKs, on neurite growth. We find that genistein greatly reduces phosphotyrosine in neurons, as expected from its action on other cells. Surprisingly, administration of genistein or lavendustin potentiated substrate-induced neurite growth in at least several different neuronal types. Stimulation of neurite growth by genistein was abolished by vanadate, providing additional evidence that inhibition of PTKs is responsible for this effect. The potentiation of growth is rather general, in that it occurs on several different extracellular matrix substrates and on two different cell adhesion molecules. Both the initiation of neurite growth and the rate of neurite elongation appear to be potentiated. Our results do not provide evidence for models of substrate-induced signal transduction that involve PTKs asa positive and necessary step, but suggest that such kinases play aregulatory role in neurite elongation. © 1992 John Wiley & Sons, Inc.     

Two forms of phosphoinositol kinase from germinating mung bean seeds

Phosphoinositol kinase, the key enzyme responsible for the biosynthesis of higher inositol phosphates has been isolated from the cotyledons of mung beans germinated for 24 hr and has been resolved into two different forms, phosphoinositol kinase A and phosphoinositol kinase B. Both forms were purified to homogeneity and characterized. The K_m values for ATP with phosphoinositol kinase A (1.78 × 10^?4 M) and phosphoinositol kinase B (3.12 × 10 ^?5 M) showed that phosphoinositol kinase B had a greater affinity for ATP. ATP could be partially replaced as phosphate donor by UTP and phosphoenolpyruvate in the case of phosphoinositol kinase A but not in the case of phosphoinositol kinase B.     

Perspectives in PDK1 evolution: Insights from photosynthetic and non-photosynthetic organisms

Protein kinases belonging to the AGC group modulate many diverse cellular processes in all eukaryotes. One important way to regulate AGC kinases is through phosphorylation by the upstream kinase PDK1. PDK1 localization and activity usually depend on interactions with phospholipids, which are mediated by a conserved lipid-binding pleckstrin homology (PH) domain. We recently analyzed putative PDK1 sequences from 17 photosynthetic organisms, finding that PDK1s from vascular and nonvascular species seem to be distinguished by the presence or absence of a PH domain, respectively. The only other reported PDK1 lacking a PH domain is from yeast (Saccharomyces cerevisiae). These observations raise questions about how plant PDK1s and their lipid-binding capabilities have evolved in relation to other eukaryotes, and what this means for PDK1 function. Here we use 100 PDK1 sequences from diverse organisms to discuss possible evolutionary aspects of plant PDK1 structure and lipid binding.