Teresa Iglesias

Identification of in Vivo Phosphorylation Sites Required for Protein Kinase D Activation

Protein kinase D (PKD) is activated by phosphorylation in intact cells stimulated by phorbol esters, cell permeant diacylglycerols, bryostatin, neuropeptides, and growth factors, but the critical activating residues in PKD have not been identified. Here, we show that substitution of Ser744 and Ser748 with alanine (PKD-S744A/S748A) completely blocked PKD activation induced by phorbol-12,13-dibutyrate (PDB) treatment of intact cells as assessed by autophosphorylation and exogenous syntide-2 peptide substrate phosphorylation assays. Conversely, replacement of both serine residues with glutamic acid (PKD-S744E/S748E) markedly increased basal activity (7.5-fold increase compared with wild type PKD). PKD-S744E/S748E mutant was only slightly further stimulated by PDB treatment in vivo, suggesting that phosphorylation of these two sites induces maximal PKD activation. Two-dimensional tryptic phosphopeptide analysis obtained from PKD mutants immunoprecipitated from 32P-labeled transfected COS-7 cells showed that two major spots present in the PDB-stimulated wild type PKD or the kinase-dead PKD-D733A phosphopeptide maps completely disappeared in the kinase-deficient triple mutant PKD-D733A/S744E/S748E. Our results indicate that PKD is activated by phosphorylation of residues Ser744 and Ser748 and thus provide the first example of a non-RD kinase that is up-regulated by phosphorylation of serine/threonine residues within the activation loop.

Protein Kinase D Activation by Mutations within Its Pleckstrin Homology Domain

Protein kinase D (PKD) is a serine/threonine protein kinase that contains a cysteine-rich repeat sequence homologous to that seen in the regulatory domain of protein kinase C (PKC) and a catalytic domain with only a low degree of sequence similarity to PKCs. PKD also contains a pleckstrin homology (PH) domain inserted between the cysteine-rich motifs and the catalytic domain that is not present in any of the PKCs. To investigate the function of the PH domain in the regulation of PKD activity, we determined the kinase activity of several PKD PH domain mutants immunoprecipitated from lysates of transiently transfected COS-7 cells. Deletion of the entire PH domain (amino acids 429–557) markedly increased the basal activity of the enzyme as assessed by autophosphorylation (∼16-fold) and exogenous syntide-2 peptide substrate phosphorylation assays (∼12-fold). Mutant PKD proteins with partial deletions or single amino acid substitutions within the PH domain (e.g. R447C and W538A) also exhibited increased basal kinase activity. These constitutive active mutants of PKD were only slightly further stimulated by phorbol-12,13-dibutyrate treatment of intact cells. Our results demonstrate, for the first time, that the PKD PH domain plays a negative role in the regulation of enzyme activity.

Spatial and temporal regulation of protein kinase D (PKD)

Protein kinase D (PKD; also known as PKCμ) is a serine/threonine kinase activated by diacylglycerol signalling pathways in a variety of cells. PKD has been described previously as Golgi‐localized, but herein we show that it is present within the cytosol of quiescent B cells and mast cells and moves rapidly to the plasma membrane after antigen receptor triggering. The membrane redistribution of PKD requires the diacylglycerol‐binding domain of the enzyme, but is independent of its catalytic activity and does not require the integrity of the pleckstrin homology domain. Antigen receptor signalling initiates in glycosphingolipid‐enriched microdomains, but membrane‐associated PKD does not co‐localize with these specialized structures. Membrane targeting of PKD is transient, the enzyme returns to the cytosol within 10 min of antigen receptor engagement. Strikingly, the membrane‐recycled PKD remains active in the cytosol for several hours. The present work thus characterizes a sustained antigen receptor‐induced signal transduction pathway and establishes PKD as a serine kinase that temporally and spatially disseminates antigen receptor signals away from the plasma membrane into the cytosol.

The pleckstrin homology domain of protein kinase D interacts preferentially with the eta isoform of protein kinase C

The results presented here demonstrate that protein kinase D (PKD) and PKCη transiently coexpressed in COS-7 cells form complexes that can be immunoprecipitated from cell lysates using specific antisera to PKD or PKCη. The presence of PKCη in PKD immune complexes was initially detected by in vitro kinase assays which reveal the presence of an 80-kDa phosphorylated band in addition to the 110-kDa band corresponding to autophosphorylated PKD. The association between PKD and PKCη was further verified by Western blot analysis and peptide phosphorylation assays that exploited the distinct substrate specificity between PKCs and PKD. By the same criteria, PKD formed complexes only very weakly with PKCε, and did not bind PKCζ. When PKCη was coexpressed with PKD mutants containing either complete or partial deletions of the PH domain, both PKCη immunoreactivity and PKC activity in PKD immunoprecipitates were sharply reduced. In contrast, deletion of an amino-terminal portion of the molecule, either cysteine-rich region, or the entire cysteine-rich domain did not interfere with the association of PKD with PKCη. Furthermore, a glutathione S-transferase-PKDPH fusion protein bound preferentially to PKCη. These results indicate that the PKD PH domain can discriminate between closely related structures of a single enzyme family, e.g. novel PKCs ε and η, thereby revealing a previously undetected degree of specificity among protein-protein interactions mediated by PH domains.

Expression of neurotrophins and the trk family of neurotrophin receptors in normal and hypothyroid rat brain

Thyroid hormone deficiency has dramatic effects on rat brain maturation. The expression of genes encoding neurotrophins and the trk family of neurotrophin receptors has been evaluated in several brain regions of normal and of neonatal or adult hypothyroid rats to analyze whether they are subject to thyroid hormone action. We found that hypothyroidism decreased trk mRNA levels in its major site of expression, the striatum, on postnatal days 5 (P5; 45%) and 15 (P15; 25%) and also in adults (35%). In contrast, no differences in trkB or trkC mRNAs levels were observed in any brain region at studied ages. According to previous reports, p75LNGFR mRNA was elevated in hypothyroid cerebellum as compared to age-matched controls on P5 and P15. We have also observed a distinct pattern for neurotrophin genes. The level of NGF mRNA was 20-50% lower in the cortex, hippocampus, and cerebellum of hypothyroid rats on neonatal hypothyroid rats on P15 and also after adult-onset hypothyroidism. Treatment of neonatally-induced hypothyroid rats with a single injection of triiodothyronine led to the recovery of hippocampal but not cortex NGF mRNA levels to that of control animals. On the contrary, no differences in the relatively high expression of the two mRNAs encoding BDNF were observed in any brain area. In contrast to a recent report, we did not find a reduction in brain NT-3 mRNA levels in hypothyroid animals. If any, the effect of thyroid deficiency in the hippocampus and cortex seems to be an early upregulation of NT-3 expression.(ABSTRACT TRUNCATED AT 250 WORDS)

Dynamic re-distribution of protein kinase D (PKD) as revealed by a GFP-PKD fusion protein: dissociation from PKD activation

Protein kinase D (PKD)/protein kinase Cμ (PKCμ), a serine/threonine protein kinase with distinct structural and enzymological properties, is rapidly activated in intact cells via PKC. The amino‐terminal region of PKD contains a cysteine‐rich domain (CRD) that directly binds phorbol esters with a high affinity. Here, we show that treatment of transfected RBL 2H3 cells with phorbol 12,13‐dibutyrate (PDB) induces a striking CRD‐dependent translocation of PKD from the cytosol to the plasma membrane, as shown by real time visualization of a functional green fluorescent protein (GFP)‐PKD fusion protein. A single amino acid substitution in the second cysteine‐rich motif of PKD (P287G) prevented PDB‐induced membrane translocation but did not affect PKD activation. Our results indicate that PKD translocation and activation are distinct processes that operate in parallel to regulate the activity and localization of this enzyme in intact cells.

Thyroid hormone-dependent transcriptional repression of neural cell adhesion molecule during brain maturation

Thyroid hormone (T3) is a main regulator of brain development acting as a transcriptional modulator. However, only a few T3‐regulated brain genes are known. Using an improved whole genome PCR approach, we have isolated seven clones encoding sequences expressed in neonatal rat brain which are under the transcriptional control of T3. Six of them, including the neural cell adhesion molecule NCAM, alpha‐tubulin and four other unidentified sequences (RBA3, RBA4, RBB3 and RBB5) were found to be upregulated in the hypothyroid brain, whereas another (RBE7) was downregulated. Binding sites for the T3 receptor (T3R/c‐erbA) were identified in the isolated clones by gel‐shift and footprinting assays. Sites in the NCAM (in an intron), alpha‐tubulin (in an exon) and RBA4 clones mediated transcriptional regulation by T3 when inserted upstream of a reporter construct. However, no effect of the NCAM clone was found when located downstream of another reporter gene. Northern blotting and in situ hybridization studies showed a higher expression of NCAM in the brain of postnatal hypothyroid rats. Since NCAM is an important morphoregulatory molecule, abnormal NCAM expression is likely to contribute to the alterations present in the brain of thyroid‐deficient humans and experimental animals.

Expression of the neurotrophin receptor trkB is regulated by the cAMP/CREB pathway in neurons

trkB as receptor for neurotrophins brain-derived neurotrophic factor (BDNF)/neurotrophin (NT)-4/5 plays a crucial role during development, maintenance of the adult brain, and its adaptation to injury or pathological conditions. In spite of this, very little is known about the mechanisms that regulate its expression. Here, we show that forskolin (Fk) rapidly stimulates the expression of both the full-length and truncated trkB isoforms in primary cultures of cortical neurons. Gel shift assays and transient transfection experiments demonstrate that this activation occurs via a protein kinase A (PKA)/cyclic AMP-responsive element-binding protein (CREB)-dependent mechanism. Activated CREB binds to the second cyclic AMP (cAMP)-responsive element (CRE) of the two CRE sites located within the P2 promoter of the trkB gene, which is able to confer cAMP responsiveness to a heterologous promoter. Our results illustrate that the trkB gene is a target for CREB regulation and explain the increase of trkB expression produced in different adaptative responses of the nervous system where CREB is participating.

Dissimilar phorbol ester binding properties of the individual cysteine‐rich motifs of protein kinase D

Protein kinase D (PKD) is a serine/threonine kinase that binds phorbol esters in a phospholipid‐dependent manner via a tandemly repeated cysteine‐rich, zinc finger‐like motif (the cysteine‐rich domain, CRD). Here, we examined whether the individual cysteine‐rich motifs of the CRD of PKD (referred to as cys1 and cys2) are functionally equivalent in mediating phorbol ester binding both in vivo and in vitro. Our results demonstrate that the cys1 and cys2 motifs of the CRD of PKD are functionally dissimilar, with the cys2 motif responsible for the majority of [3H]phorbol 12,13‐dibutyrate (PDB) binding, both in vivo and in vitro.

Phosphorylation-dependent protein kinase Dactivation

The novel mouse serine‐threonine kinase protein kinase D (PKD) is activated in intact Swiss 3T3 cells stimulated by phorbol esters, cell permeant diacylglycerols, bryostatin, neuropeptides and growth factors via a phosphorylation‐dependent mechanism requiring protein kinase C (PKC) activity. Structural comparison of the PKD catalytic domain with other kinases reveals a close similarity with MEK familiy kinases, which are activated upon phosphorylation of key serine and threonine residues in a region termed the activation loop. To study the regulation of PKD, we transfected mutant PKD cDNAs in which putative activation loop serine residues 744 and 748 were mutated to either alanine or glutamic acid into COS‐7 cells. Replacement of serines 744 and 748 with alanine prevented activation of the overexpressed PKD form upon phorbol ester treatment of cells, whereas replacement with glutamic acid results in full constitutive activation. Single serine to glutamic acid replacement mutants were partially activated. In vivo 32P‐labeling and two‐dimensional phosphopeptide mapping of PKD and catalytically inactive PKD mutants at serine 744, 748 or at both residues revealed that phorbol ester‐sensitive phosphopeptides could be selectively eliminated from pattrens observed as a result of these mutations. Treatment of cells with the PKC inhibitor GFI also prevented the appearance of phosphopeptide spots occuring in response to phorbol ester stimulation. These results provide direct evidence that PKD becomes activated in vivo as a consequence of PKC‐mediated phosphorylation of serines 744 and 748. These results support our view of PKD as an important clement in PKC signal transduction.