Lipid raft disruption triggers protein kinase C and Src-dependent protein kinase D activation and Kidins220 phosphorylation in neuronal cells

Kidins220 (kinase D-interacting substrate of 220 kDa) is a novel neurospecific protein recently cloned as the first substrate for the Ser/Thr kinase protein kinase D (PKD). Herein we report that Kidins220 is constitutively associated to lipid rafts in PC12 cells, rat primary cortical neurons, and brain synaptosomes. Immunocytochemistry and confocal microscopy together with sucrose gradient fractionation show co-localization of Kidins220 and lipid raft-associated proteins. In addition, cholesterol depletion of cell membranes with methyl-beta-cyclodextrin dramatically alters Kidins220 localization and detergent solubility. By studying the putative involvement of lipid rafts in PKD activation and signaling we have found that active PKD partitions in lipid raft fractions after sucrose gradient centrifugation and that green fluorescent protein-PKD translocates to lipid raft microdomains at the plasma membrane after phorbol ester treatment. Strikingly, lipid rafts disruption by methyl-beta-cyclodextrin delays green fluorescent protein-PKD translocation, as determined by live cell confocal microscopy, and activates PKD, increasing Kidins220 phosphorylation on Ser(919) by a mechanism involving PKCepsilon and the small soluble tyrosine kinase Src. Collectively, these results reveal the importance of lipid rafts on PKD activation, translocation, and downstream signaling to its substrate Kidins220.     

A phosphorylation state-specific antibody recognizes Hsp27, a novel substrate of protein kinase D

The use of phosphorylation state-specific antibodies has revolutionized the field of cellular signaling by Ser/Thr protein kinases. A more recent application of this technology is the development of phospho-specific antibodies that specifically recognize the consensus substrate phosphorylated motif of a given protein kinase. Here, we describe the development and use of such an antibody which is directed against the optimal phosphorylation motif of protein kinase D (PKD). A degenerate phosphopeptide library with fixed residues corresponding to the consensus LXR(Q/K/E/M)(M/L/K/E/Q/A)S*XXXX was used as an antigen to generate an antibody that recognizes this motif. We characterized the antibody by enzyme-linked immunosorbent assay and with immobilized peptide arrays and also detected immunoreactive phosphoproteins in HeLa cells stimulated with agonists known to activate PKD. Silencing PKD expression using RNA interference validated the specificity of this antibody immunoreactive against putative substrates. The antibody also detected the PKD substrates RIN1 and HDAC5. Knowledge of the PKD consensus motif also enabled us to identify Ser(82) in the human heat shock protein Hsp27 as a novel substrate for PKD. We term this antibody anti-PKD pMOTIF and predict that it will enable the discovery of novel PKD substrate proteins in cells.     

Kidins220/ARMS interacts with Pdzrn3, a protein containing multiple binding domains

We report the identification of a novel partner of Kidins220/ARMS (Kinase D-interacting substrate of 220 kDa/Ankyrin Repeat-rich Membrane Spanning) an adaptor of neurotrophin receptors playing crucial roles during neurogenesis. Screening a phage display library of brain cDNA products we found that D. rerio Pdzrn3, a protein containing RING-finger and PDZ-domains, interacts with Kidins220/ARMS through its first PDZ-domain. Both zebrafish proteins share high homology with the corresponding mammalian proteins and both genes are developmentally expressed in neural districts where early neurogenesis occurs. The interaction was also confirmed by biochemical assays and by co-localization at the tips of growing neurites of PC12 cells induced with nerve growth factor.     

Ca2+/calmodulin-dependent protein kinase enriched in cerebellar granule cells. Identification of a novel neuronal calmodulin-dependent protein kinase

Ca2+-sensitive protein kinases are thought to play a pivotal role in Ca2+-mediated neuronal communication. We describe here the cloning, purification, and characterization of a major Ca2+/calmodulin-dependent, brain-specific protein kinase which is particularly enriched in cerebellar granule cells. The enzyme is comprised of Mr 65,000 and 67,000 polypeptides which copurify to homogeneity and phosphorylate synapsin I. The protein kinase is coded for by two poly(A+) RNAs of 2.0 and 3.5 kilobases which probably derive from a single gene. Two cDNA inserts, one of 198 base pairs and one of 1225 base pairs, contain a total of 677 base pairs of the protein coding sequence which includes sequences homologous to other calmodulin-dependent protein kinases including part of the calmodulin-binding domain. The surprising presence of extended sequences which are enriched in glutamate residues may influence the subcellular distribution of this kinase. Immunohistochemical localization with an affinity-purified antibody reveals that whereas the enzyme is expressed in several neuronal subpopulations, it is exceptionally enriched in the granule cells of the cerebellum. The relevance of the biochemical, molecular, and histologic properties of this enzyme is discussed in the context of neuronal Ca2+ signaling.     

Identification of a novel substrate for TNFalpha-induced kinase NUAK2

TNFα has multiple important cellular functions both in normal cells and in tumor cells. To explore the role of TNFα, we identified NUAK family, SNF1-like kinase 2 (NUAK2), as a TNFα-induced kinase by gene chip analysis. NUAK2 is known to be induced by various cellular stresses and involved in cell mortality, however, its substrate has never been identified. We developed original protocol of de novo screening for kinase substrates using an in vitro kinase assay and high performance liquid chromatography (HPLC). Using this procedure, we identified myosin phosphatase target subunit 1 (MYPT1) as a specific substrate for NUAK2. MYPT1 was phosphorylated at another site(s) by NUAK2, other than known Rho-kinase phosphorylation sites (Thr696 or Thr853) responsible for inhibition of myosin phosphatase activity. These data suggests different phosphorylation and regulation of MYPT1 activity by NUAK2.     

Characterization of EVL-I as a protein kinase D substrate

EVL-I is a splice variant of EVL (Ena/VASP like protein), whose in vivo function and regulation are still poorly understood. We found that Protein Kinase D (PKD) interacts in vitro and in vivo with EVL-I and phosphorylates EVL-I in a 21 amino acid alternately-included insert in the EVH2 domain. Following knockdown of the capping protein CPβ and spreading on laminin, phosphorylated EVL-I can support filopodia formation and the phosphorylated EVL-I is localized at filopodial tips. Furthermore, we found that the lamellipodial localization of EVL-I is unaffected by phosphorylation, but that impairment of EVL-I phosphorylation is associated with ruffling of lamellipodia upon PDBu stimulation. Besides the lamellipodial and filopodial localization of phosphorylated EVL-I in fibroblasts, we determined that EVL-I is hyperphosphorylated and localized in the cell–cell contacts of certain breast cancer cells and mouse embryo keratinocytes. Taken together, our results show that phosphorylated EVL-I is present in lamellipodia, filopodia and cell–cell contacts and suggest the existence of signaling pathways that may affect EVL-I via phosphorylation of its EVH2 domain.     

Identification and characterization of a novel testis-specific gene CKT2, which encodes a substrate for protein kinase CK2

Protein kinase CK2 is a serine/threonine kinase known to phosphorylate numerous substrates. CK2 is implicated in several physiologic and pathologic processes, particularly in cancer biology. CK2 is comprised of several subunits, including CK2α, CK2α′ and CK2β. Inactivation of CK2α′ leads to chromatin degeneration of germ cells, resulting in male sterility. To identify additional targets of CK2α′ in testes and to determine the role of CK2α′ in germ cell nuclear integrity, GST pull-down and protein–protein interaction assays were conducted. A novel testis-specific gene, CKT2 (CK2 Target protein 2), was found whose product interacts with and is phosphorylated by CK2 in vitro and in vivo. CKT2 is a 30.2 kDa protein with one coiled-coil domain and six putative phosphorylation sites. High expression of CKT2 correlated with chromatin condensation of spermatids in murine testes. Findings reported herein demonstrate that CKT2 is a target protein of native CK2α′ in testes and suggest that CKT2 plays a role in chromatin regulation of male germ cells.     

KIBRA is a novel substrate for protein kinase Czeta

WW domain-containing proteins are found in all eukaryotic cells and they are involved in the regulation of a wide variety of cellular functions. We recently identified the neuronal protein KIBRA as novel member of this family of signal transducers. In this report, we describe the identification of protein kinase C (PKC) zeta as a KIBRA-interacting protein. PKCzeta is known to play an important role in synaptic plasticity and memory formation but its specific targets are not well known. Our studies presented here revealed that KIBRA is a novel substrate for PKCzeta and suggest that PKCzeta phosphorylation may regulate the cellular function of KIBRA.     

Identification of a substrate for Pkn2, a protein Ser/Thr kinase from Myxococcus xanthus by a novel method for substrate identification

Eukaryotic cells contain a large number of protein Ser/ Thr kinases, which play important roles in signal transduction required for cell proliferation, differentiation, and stress response and adaptation. It is also known that some prokaryotes contain a family of protein Ser/Thr kinases. A major challenge in the characterization of these kinases is how to identify their specific substrates. Here we developed such a method using a protein Ser/Thr kinase, Pkn2 from Myxococcus xanthus, a Gram-negative soil bacterium. When Pkn2 is inducibly expressed in E. coli, cells are unable to form colonies on agar plates. This lethal effect of Pkn2 was eliminated in an inactive Pkn2 mutant in which the highly conserved Lys residue was changed to Asn, indicating that phosphorylation of a cellular protein(s) in E. coli resulted in growth arrest. Several clones from an E. coli genomic library were found to suppress the lethal effect when co-expressed with pkn2. Four out of seven multi-copy suppressors were identified to encode HU, (3 for HUalpha and 1 for HUB) a histone-like DNA binding protein. Purified HUalpha was found to be specifically phosphorylated by Pkn2 at Thr-59, and the phosphorylated HUalpha became unable to bind to DNA, suggesting that the phosphorylation of endogenous HU proteins by Pkn2 contributed at least in part to the lethal effect in E. coli. The present method termed the STEK method (Suppressors of Toxic Effects of Kinases) may be widely used for the substrate identification not only for prokaryotic protein Ser/Thr kinases but also for eukaryotic kinases.     

Kidins220/ARMS is a novel modulator of short-term synaptic plasticity in hippocampal GABAergic neurons

Kidins220 (Kinase D interacting substrate of 220 kDa)/ARMS (Ankyrin Repeat-rich Membrane Spanning) is a scaffold protein highly expressed in the nervous system. Previous work on neurons with altered Kidins220/ARMS expression suggested that this protein plays multiple roles in synaptic function. In this study, we analyzed the effects of Kidins220/ARMS ablation on basal synaptic transmission and on a variety of short-term plasticity paradigms in both excitatory and inhibitory synapses using a recently described Kidins220 full knockout mouse. Hippocampal neuronal cultures prepared from embryonic Kidins220(-/-) (KO) and wild type (WT) littermates were used for whole-cell patch-clamp recordings of spontaneous and evoked synaptic activity. Whereas glutamatergic AMPA receptor-mediated responses were not significantly affected in KO neurons, specific differences were detected in evoked GABAergic transmission. The recovery from synaptic depression of inhibitory post-synaptic currents in WT cells showed biphasic kinetics, both in response to paired-pulse and long-lasting train stimulation, while in KO cells the respective slow components were strongly reduced. We demonstrate that the slow recovery from synaptic depression in WT cells is caused by a transient reduction of the vesicle release probability, which is absent in KO neurons. These results suggest that Kidins220/ARMS is not essential for basal synaptic transmission and various forms of short-term plasticity, but instead plays a novel role in the mechanisms regulating the recovery of synaptic strength in GABAergic synapses.     

Identification of a new in vitro substrate of tyrosine protein kinase

Recent studies in our laboratory [Tokuda, M., Khanna, N.C., Aurora, A., & Waisman, D. M. (1986) Biochem. Biophys. Res. Commun. 139, 910-917] have identified in membranes of rat spleen two tyrosine protein kinases named TPK-I and TPK-II. In this paper the identification of the Ca2+ binding protein CAB-48 as a major in vitro substrate of TPK-II is reported. TPK-II catalyzed the incorporation of 0.73 mol of phosphate/mol of CAB-48. Phosphoamino acid analysis revealed that phosphorylation of CAB-48 was specific for tyrosine residues. Phosphorylation of CAB-48 by TPK-I (rat spleen), protein kinase C, casein kinase I, casein kinase II, cAMP-dependent protein kinase, or calcium calmodulin dependent protein kinase was not observed.     

Identification of protein kinase D as a novel contraction-activated kinase linked to GLUT4-mediated glucose uptake, independent of AMPK

Contraction-induced glucose uptake is only partly mediated by AMPK activation. We examined whether the diacylglycerol-sensitive protein kinase D (PKD; also known as novel PKC isoform mu) is also involved in the regulation of glucose uptake in the contracting heart. As an experimental model, we used suspensions of cardiac myocytes, which were electrically stimulated to contract or treated with the contraction-mimicking agent oligomycin. Induction of contraction at 4 Hz in cardiac myocytes or treatment with 1 microM oligomycin enhanced (i) autophosphorylation of PKD at Ser916 by 5.1- and 3.8-fold, respectively, (ii) phosphorylation of PKD's downstream target cardiac-troponin-I (cTnI) by 2.9- and 2.1-fold, respectively, and (iii) enzymatic activity of immunoprecipitated PKD towards the substrate peptide syntide-2 each by 1.5-fold. Although AMPK was also activated under these same conditions, in vitro phosphorylation assays and studies with cardiac myocytes from AMPKalpha2(-/-) mice indicated that activation of PKD occurs independent of AMPK activation. CaMKKbeta, and the cardiac-specific PKC isoforms alpha, delta, and epsilon were excluded as upstream kinases for PKD in contraction signaling because none of these kinases were activated by oligomycin. Stimulation of glucose uptake and induction of GLUT4 translocation in cardiac myocytes by contraction and oligomycin each were sensitive to inhibition by the PKC/PKD inhibitors staurosporin and calphostin-C. Together, these data elude to a role of PKD in contraction-induced GLUT4 translocation. Finally, the combined actions of PKD on cTnI phosphorylation and on GLUT4 translocation would efficiently link accelerated contraction mechanics to increased energy production when the heart is forced to increase its contractile activity.     

Purification, cloning, and characterization of Nek8, a novel NIMA-related kinase, and its candidate substrate Bicd2

We describe the isolation, cloning, and characterization of human Nek8, a new mammalian NIMA-related kinase, and its candidate substrate Bicd2. Nek8 was isolated as a beta-casein kinase activity in rabbit lung and has an N-terminal catalytic domain homologous to the Nek family of protein kinases. Nek8 also contains a central domain with homology to RCC1, a guanine nucleotide exchange factor for the GTPase Ran, and a C-terminal coiled-coil domain. Like Nek2, Nek8 prefers beta-casein over other exogenous substrates, has shared biochemical requirements for kinase activity, and is capable of autophosphorylation and oligomerization. Nek8 activity is not cell cycle regulated, but like Nek3, levels are consistently higher in G(0)-arrested cells. During the purification of Nek8 a second protein co-chromatographed with Nek8 activity. This protein, Bicd2, is a human homolog of the Drosophila protein Bicaudal D, a coiled-coil protein. Bicd2 is phosphorylated by Nek8 in vitro, and the endogenous proteins associate in vivo. Bicd2 localizes to cytoskeletal structures, and its subcellular localization is dependent on microtubule morphology. Treatment of cells with nocodazole leads to dramatic reorganization of Bicd2, and correlates with Nek8 phosphorylation. This may be indicative of a role for Nek8 and Bicd2 associated with cell cycle independent microtubule dynamics.     

Identification of phospholipase C-gamma1 as a mitogen-activated protein kinase substrate

The discovery of sequence motifs that mediate protein-protein interactions, coupled with the availability of protein amino acid sequence data, allows for the identification of putative protein binding pairs. The present studies were based on our identification of an amino acid sequence in phosphatidylinositol-specific phospholipase C-gamma1 (PLC-gamma1) that fits the consensus sequence for a mitogen-activated protein kinase (MAPK) binding site, termed the D-domain. Extracellular signal-regulated kinase 2 (ERK2), an MAPK, and phospho-ERK2 were bound by an immobilized peptide sequence containing the identified PLC-gamma1 D-domain. Furthermore, a peptide containing the PLC-gamma1 D-domain was able to competitively inhibit the in vitro phosphorylation of recombinant PLC-gamma1 by recombinant phospho-ERK2, whereas a control peptide derived from a distant region of PLC-gamma1 was ineffective. Similarly, the peptide containing the PLC-gamma1 D-domain, but not the control peptide, competitively inhibited the in vitro phosphorylation of Elk-1 and c-Jun catalyzed by recombinant phospho-ERK2 and phospho-c-Jun N-terminal kinase 3 (phospho-JNK3), another type of MAPK, respectively. Incubation of anti-PLC-gamma1 immunocomplexes isolated from rat brain with recombinant phospho-ERK2 opposed the increase in PLC-gamma1-catalyzed hydrolysis of phosphatidylinositol 4,5-P(2) (PtdIns(4,5)P(2)), which was produced by a tyrosine kinase associated with the immunocomplexes, whereas in vitro phosphorylation of recombinant PLC-gamma1 by recombinant phospho-ERK2 did not alter PLC-gamma1-catalyzed PtdIns(4,5)P(2) hydrolysis. These studies have uncovered a previously unidentified mechanism for the integration of PLC-gamma1- and ERK2-dependent signaling.     

Properties of p19, a novel cAMP-dependent protein kinase substrate protein purified from bovine brain

We report the purification from bovine brain and describe some of the properties of a 19-kDa protein, p19, which we have previously shown to undergo hormone-dependent, cAMP-mediated phosphorylation in several peptide hormone-producing tumor cells. The procedure for purifying p19 to apparent homogeneity utilized ammonium sulfate fractionation, sequential chromatography on DEAE-cellulose and phenyl-Sepharose, followed by fast protein liquid chromatography using a Mono Q and, finally, a C8 reverse-phase column. The yield was 0.3-0.5 mg of p19/kg of brain. The molecular weight (Mr = 19,000) and frictional ratio (f/f0 = 1.87) of p19, which were derived from its Stokes radius (33 A) and sedimentation constant (s20,w = 1.4), suggest that the native form of p19 is an asymmetrically shaped monomer. We provide evidence to suggest that p19 is isolated as a mixture of molecular forms consisting of an unphosphorylated form and of three phosphoforms indicative of multisite phosphorylation. These forms cosedimented on sucrose density gradients and coeluted on gel filtration, hydrophobic chromatography, and reverse-phase fast protein liquid chromatography. They were resolved from each other by anion-exchange chromatography. The unphosphorylated form (pI 6.2) was phosphorylated by catalytic subunit of cAMP-dependent protein kinase to a stoichiometry of 0.5 mol of P/mol of p19, thereby giving rise to the three phosphoforms (pI 5.8, pI 5.6, and pI 5.2, respectively). We conclude that p19 is a novel cAMP-dependent protein kinase substrate protein that is present in brain and in peptide hormone-producing tumor cells. Its function remains to be identified.     

Identification of a novel annexin in Hydra vulgaris. Characterization, cDNA cloning, and protein kinase C phosphorylation of annexin XII.

As a first step toward the elucidation of a simple animal model in which to investigate annexin function, we identified, isolated, and characterized a novel annexin from Hydra vulgaris, annexin XII. A hydra cDNA library was screened using a probe generated by polymerase chain reaction from primers based on the partial amino acid sequence of annexin XII. Annexin XII cDNA was cloned and the functional protein was expressed in high yields in Escherichia coli. The annexin XII cDNA sequence predicted a 316-amino acid protein that had between 44 and 54% sequence identity with the Ca2+-binding core domains of previously characterized vertebrate and Drosophila annexins. The amino-terminal domain of annexin XII did not have sequence similarity with other known annexins except at and around a site that resembled known protein kinase C (PKC) phosphorylation sites in other annexins. As anticipated from its sequence, annexin XII was a high affinity substrate for purified rat brain PKC; half-maximal phosphorylation occurred below 0.1 microM annexin XII, and incorporation of up to 0.8 mol of phosphate/mol of annexin XII was observed. A PKC-like activity in hydra extracts also phosphorylated annexin XII. In summary, hydra promises to be a valuable model system for investigating the biological function of annexins and for determining how this function is modulated by PKC phosphorylation.