Engineered synaptic tools reveal localized cAMP signaling in synapse assembly

2021 ◽  
Vol 221 (2) ◽  
Author(s):  
Richard Sando ◽  
Milan Lyndie Ho ◽  
Xinran Liu ◽  
Thomas C. Südhof
Keyword(s):  
Synapse Formation ◽  
Camp Signaling ◽  
Protein Kinase Activity ◽  
Physiological Mechanisms ◽  
Ca1 Neurons ◽  
Synaptic Targeting ◽  
Dependent Protein ◽  
Virus Tracing ◽  
Camp Levels

The physiological mechanisms driving synapse formation are elusive. Although numerous signals are known to regulate synapses, it remains unclear which signaling mechanisms organize initial synapse assembly. Here, we describe new tools, referred to as “SynTAMs” for synaptic targeting molecules, that enable localized perturbations of cAMP signaling in developing postsynaptic specializations. We show that locally restricted suppression of postsynaptic cAMP levels or of cAMP-dependent protein-kinase activity severely impairs excitatory synapse formation without affecting neuronal maturation, dendritic arborization, or inhibitory synapse formation. In vivo, suppression of postsynaptic cAMP signaling in CA1 neurons prevented formation of both Schaffer-collateral and entorhinal-CA1/temporoammonic-path synapses, suggesting a general principle. Retrograde trans-synaptic rabies virus tracing revealed that postsynaptic cAMP signaling is required for continuous replacement of synapses throughout life. Given that postsynaptic latrophilin adhesion-GPCRs drive synapse formation and produce cAMP, we suggest that spatially restricted postsynaptic cAMP signals organize assembly of postsynaptic specializations during synapse formation.

Relaxing effect of pharmacologic interventions increasing cAMP in rat heart

1981 ◽  
Vol 240 (4) ◽  
pp. H441-H447
Author(s):  
L. Vittone ◽  
A. Grassi ◽  
L. Chiappe ◽  
M. Argel ◽  
H. E. Cingolani
Keyword(s):  
Protein Kinase ◽  
Rat Heart ◽  
Kinase Activity ◽  
Isolated Rat Heart ◽  
Protein Kinase Activity ◽  
Dependent Protein Kinase ◽  
Camp Dependent Protein Kinase ◽  
High Calcium ◽  
Dependent Protein ◽  
Camp Levels

The relationship between cAMP and relaxation was studied in the isolated rat heart beating at constant rate and perfused at constant coronary flow. After treatment during 1 min with different positive inotropic interventions, cyclic nucleotide levels (cAMP and cGMP) and cAMP-dependent protein kinase activity were determined in heart homogenates. Glucagon, norepinephrine, and isoproterenol increased cAMP from 0.503 +/- 0.025 pmol/mg wet wt to 1.051 +/- 0.099, 0.900 +/- 0.064, and 0.982 +/- 0.138, respectively. Simultaneously glucagon, norepinephrine, and isoproterenol increased cAMP-dependent protein kinase activity ratio from 0.21 +/- 0.02 to 0.45 +/- 0.04, 0.33 +/- 0.02, and 0.34 +/- 0.02, respectively. The ratio between maximal velocities of contraction and relaxation (+T/-T) was significantly decreased by these interventions, whereas time to peak tension (TTP) was shortened by norepinephrine and isoproterenol. High calcium, ouabain, and paired stimulation did not affect cAMP levels, TTP, or +T/-T. A striking correlation was found between cAMP-dependent protein kinase activity and relaxation induces, i.e., TTP, -T, or +T/-T (r = +/- 0.7 to -0.9). Results suggest that inotropic interventions increasing cAMP levels might be primarily affecting intracellular mechanisms causing relaxation.

scholarly journals NCAM promotes assembly and activity-dependent remodeling of the postsynaptic signaling complex

2006 ◽  
Vol 174 (7) ◽  
pp. 1071-1085 ◽  
Author(s):  
Vladimir Sytnyk ◽  
Iryna Leshchyns'ka ◽  
Alexander G. Nikonenko ◽  
Melitta Schachner
Keyword(s):  
Nmda Receptor ◽  
Synapse Formation ◽  
Long Term Potentiation ◽  
Receptor Activation ◽  
Synaptic Strength ◽  
Synaptic Targeting ◽  
Signaling Complex ◽  
Term Potentiation ◽  
Neural Cell Adhesion ◽  
Dependent Protein

The neural cell adhesion molecule (NCAM) regulates synapse formation and synaptic strength via mechanisms that have remained unknown. We show that NCAM associates with the postsynaptic spectrin-based scaffold, cross-linking NCAM with the N-methyl-d-aspartate (NMDA) receptor and Ca2+/calmodulin-dependent protein kinase II α (CaMKIIα) in a manner not firmly or directly linked to PSD95 and α-actinin. Clustering of NCAM promotes formation of detergent-insoluble complexes enriched in postsynaptic proteins and resembling postsynaptic densities. Disruption of the NCAM–spectrin complex decreases the size of postsynaptic densities and reduces synaptic targeting of NCAM–spectrin–associated postsynaptic proteins, including spectrin, NMDA receptors, and CaMKIIα. Degeneration of the spectrin scaffold in NCAM-deficient neurons results in an inability to recruit CaMKIIα to synapses after NMDA receptor activation, which is a critical process in NMDA receptor–dependent long-term potentiation. The combined observations indicate that NCAM promotes assembly of the spectrin-based postsynaptic signaling complex, which is required for activity-associated, long-lasting changes in synaptic strength. Its abnormal function may contribute to the etiology of neuropsychiatric disorders associated with mutations in or abnormal expression of NCAM.

scholarly journals Phosphorylation of the inhibitory subunit of troponin in perfused hearts of mice deficient in phosphorylase kinase. Evidence for the phosphorylation of troponin by adenosine 3′:5′-phosphate-dependent protein kinase in vivo

1977 ◽  
Vol 168 (2) ◽  
pp. 307-310 ◽  
Author(s):  
P J England
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Kinase Activity ◽  
Troponin I ◽  
Protein Kinase Activity ◽  
Phosphorylase Kinase ◽  
Dependent Protein Kinase ◽  
Parallel Increase ◽  
Dependent Protein

When hearts from control and phosphorylase kinase-deficient (I strain) mice were perfused with 0.1 micrometer-DL-isoprenaline, there was a parallel increase in contraction, cyclic AMP concentration and troponin I phosphorylation. However, there was no increase in phosphorylase a in the I-strain hearts, whereas the control hearts showed a large increase. Assays of I-strain heart extracts showed a normal cyclic AMP-dependent protein kinase activity but no phosphorylase kinase activity. It is concluded that troponin I is phosphorylated in intact hearts by protein kinase and not phosphorylase kinase.

scholarly journals Latrophilin GPCR Signaling Mediates Synapse Formation

2021 ◽  
Author(s):  
Richard Sando ◽  
Thomas C. Südhof
Keyword(s):  
Synapse Formation ◽  
Neural Circuit ◽  
Gpcr Signaling ◽  
Synapse Loss ◽  
Ca1 Region ◽  
In Trans ◽  
Synaptic Complexes ◽  
Camp Levels ◽  
The Brain

ABSTRACTNeural circuit assembly in the brain requires precise establishment of synaptic connections, but the mechanisms of synapse assembly remain incompletely understood. Latrophilins are postsynaptic adhesion-GPCRs that engage in trans-synaptic complexes with presynaptic teneurins and FLRTs. In CA1-region neurons, Latrophilin-2 and Latrophilin-3 are essential for formation of entorhinal-cortex-derived and Schaffer-collateral-derived synapses, respectively. However, it is unknown whether latrophilins function as GPCRs in synapse formation. Here, we show that Latrophilin-2 and Latrophilin-3 exhibit constitutive GPCR activity that increases cAMP levels, which was blocked by a mutation interfering with G-protein and arrestin interactions of GPCRs. The same mutation impaired the ability of Latrophilin-2 and Latrophilin-3 to rescue the synapse-loss phenotype in Latrophilin-2 and Latrophilin-3 knockout neurons in vivo. Our results suggest that Latrophilin-2 and Latrophilin-3 require GPCR signaling in synapse formation, indicating that latrophilins promote synapse formation in the hippocampus by activating a classical GPCR-signaling pathway.

scholarly journals The DNA-Dependent Protein Kinase Catalytic Subunit Is Phosphorylated In Vivo on Threonine 3950, a Highly Conserved Amino Acid in the Protein Kinase Domain

2006 ◽  
Vol 27 (5) ◽  
pp. 1581-1591 ◽  
Author(s):  
Pauline Douglas ◽  
Xiaoping Cui ◽  
Wesley D. Block ◽  
Yaping Yu ◽  
Shikha Gupta ◽  
...  
Keyword(s):  
Amino Acid ◽  
Protein Kinase ◽  
Kinase Domain ◽  
Catalytic Subunit ◽  
Protein Kinase Activity ◽  
Dependent Protein Kinase ◽  
Protein Kinase Domain ◽  
Dependent Protein

ABSTRACT The protein kinase activity of the DNA-dependent protein kinase (DNA-PK) is required for the repair of DNA double-strand breaks (DSBs) via the process of nonhomologous end joining (NHEJ). However, to date, the only target shown to be functionally relevant for the enzymatic role of DNA-PK in NHEJ is the large catalytic subunit DNA-PKcs itself. In vitro, autophosphorylation of DNA-PKcs induces kinase inactivation and dissociation of DNA-PKcs from the DNA end-binding component Ku70/Ku80. Phosphorylation within the two previously identified clusters of phosphorylation sites does not mediate inactivation of the assembled complex and only partially regulates kinase disassembly, suggesting that additional autophosphorylation sites may be important for DNA-PK function. Here, we show that DNA-PKcs contains a highly conserved amino acid (threonine 3950) in a region similar to the activation loop or t-loop found in the protein kinase domain of members of the typical eukaryotic protein kinase family. We demonstrate that threonine 3950 is an in vitro autophosphorylation site and that this residue, as well as other previously identified sites in the ABCDE cluster, is phosphorylated in vivo in irradiated cells. Moreover, we show that mutation of threonine 3950 to the phosphomimic aspartic acid abrogates V(D)J recombination and leads to radiation sensitivity. Together, these data suggest that threonine 3950 is a functionally important, DNA damage-inducible phosphorylation site and that phosphorylation of this site regulates the activity of DNA-PKcs.

scholarly journals Toxoplasma gondiiAttachment to Host Cells Is Regulated by a Calmodulin-like Domain Protein Kinase

2001 ◽  
Vol 276 (15) ◽  
pp. 12369-12377 ◽  
Author(s):  
Heidi Kieschnick ◽  
Therese Wakefield ◽  
Carl Anthony Narducci ◽  
Con Beckers
Keyword(s):  
Protein Kinase ◽  
Protein Kinases ◽  
Kinase Activity ◽  
Host Cells ◽  
Protein Kinase Activity ◽  
Calcium Dependent ◽  
Dependent Protein ◽  
Calcium Dependent Protein Kinases ◽  
Domain Protein

The role of calcium-dependent protein kinases in the invasion ofToxoplasma gondiiinto its animal host cells was analyzed. KT5926, an inhibitor of calcium-dependent protein kinases in other systems, is known to block the motility ofToxoplasmatachyzoites and their attachment to host cells.In vivo, KT5926 blocks the phosphorylation of only three parasite proteins, and in parasite extracts only a single KT5926-sensitive protein kinase activity was detected. This activity was calcium-dependent but did not require calmodulin. In a search for calcium-dependent protein kinases inToxoplasma, two members of the class of calmodulin-like domain protein kinases (CDPKs) were detected. TgCDPK2 was only expressed at the mRNA level in tachyzoites, but no protein was detected. TgCDPK1 protein was expressed inToxoplasmatachyzoites and cofractionated precisely with the peak of KT5926-sensitive protein kinase activity. TgCDPK1 kinase activity was calcium-dependent but did not require calmodulin or phospholipids. TgCDPK1 was found to be inhibited effectively by KT5926 at concentrations that block parasite attachment to host cells.In vitro, TgCDPK1 phosphorylated three parasite proteins that migrated identical to the three KT5926-sensitive phosphoproteins detectedin vivo. Based on these observations, a central role is suggested for TgCDPK1 in regulatingToxoplasmamotility and host cell invasion.

scholarly journals An inhibitor of yeast cyclin-dependent protein kinase plays an important role in ensuring the genomic integrity of daughter cells.

1994 ◽  
Vol 14 (5) ◽  
pp. 3320-3328 ◽  
Author(s):  
T T Nugroho ◽  
M D Mendenhall
Keyword(s):  
Protein Kinase ◽  
Protein Kinase Activity ◽  
Gene Products ◽  
Dependent Protein Kinase ◽  
Gene Encoding ◽  
Daughter Cells ◽  
G2 Checkpoint ◽  
Dependent Protein ◽  
Segregation Of Chromosomes

The gene encoding a 40-kDa protein, previously studied as a substrate and inhibitor of the yeast cyclin-dependent protein kinase, Cdc28, has been cloned. The DNA sequence reveals that p40 is a highly charged protein of 32,187 Da with no significant homology to other proteins. Overexpression of the gene encoding p40, SIC1, produces cells with an elongated but morphology similar to that of cells with depleted levels of the CLB gene products, suggesting that p40 acts as an inhibitor of Cdc28-Clb complexes in vivo. A SIC1 deletion is viable and has highly increased frequencies of broken and lost chromosomes. The deletion strain segregates out many dead cells that are primarily arrested at the G2 checkpoint in an asymmetric fashion. Only daughters and young mothers display the lethal defect, while experienced mothers appear to grow normally. These results suggest that negative regulation of Cdc28 protein kinase activity by p40 is important for faithful segregation of chromosomes to daughter cells.

scholarly journals Identification of in vitro and in vivo phosphorylation sites in the catalytic subunit of the DNA-dependent protein kinase

2002 ◽  
Vol 368 (1) ◽  
pp. 243-251 ◽  
Author(s):  
Pauline DOUGLAS ◽  
Gopal P. SAPKOTA ◽  
Nick MORRICE ◽  
Yaping YU ◽  
Aaron A. GOODARZI ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Okadaic Acid ◽  
Kinase Activity ◽  
Catalytic Subunit ◽  
Protein Sequencing ◽  
Protein Kinase Activity ◽  
Dependent Protein Kinase ◽  
Dependent Protein

The DNA-dependent protein kinase (DNA-PK) is required for the repair of DNA double-strand breaks (DSBs), such as those caused by ionizing radiation and other DNA-damaging agents. DNA-PK is composed of a large catalytic subunit (DNA-PKcs) and a heterodimer of Ku70 and Ku80 that assemble on the ends of double-stranded DNA to form an active serine/threonine protein kinase complex. Despite in vitro and in vivo evidence to support an essential role for the protein kinase activity of DNA-PK in the repair of DNA DSBs, the physiological targets of DNA-PK have remained elusive. We have previously shown that DNA-PK undergoes autophosphorylation in vitro, and that autophosphorylation correlates with loss of protein kinase activity and dissociation of the DNA-PK complex. Also, treatment of cells with the protein phosphatase inhibitor, okadaic acid, enhances DNA-PKcs phosphorylation and reduces DNA-PK activity in vivo. Here, using solid-phase protein sequencing, MS and phosphospecific antibodies, we have identified seven in vitro autophosphorylation sites in DNA-PKcs. Six of these sites (Thr2609, Ser2612, Thr2620, Ser2624, Thr2638 and Thr2647) are clustered in a region of 38 amino acids in the central region of the protein. Five of these sites (Thr2609, Ser2612, Thr2638, Thr2647 and Ser3205) are conserved between six vertebrate species. Moreover, we show that DNA-PKcs is phosphorylated in vivo at Thr2609, Ser2612, Thr2638 and Thr2647 in okadaic acid-treated human cells. We propose that phosphorylation of these sites may play an important role in DNA-PK function.

Multiple sites for interaction of prostaglandin and vasopressin in toad urinary bladder

1981 ◽  
Vol 241 (6) ◽  
pp. F625-F631
Author(s):  
D. Schlondorff ◽  
C. P. Carvounis ◽  
M. Jacoby ◽  
J. A. Satriano ◽  
S. D. Levine
Keyword(s):  
Water Flow ◽  
Protein Kinase Activity ◽  
Toad Urinary Bladder ◽  
Dependent Protein Kinase ◽  
Camp Content ◽  
Camp Generation ◽  
Dependent Protein ◽  
Endogenous Prostaglandins ◽  
Camp Levels ◽  
Inhibited Water

The interaction of vasopressin with prostaglandins were examined in the toad bladder by determining water flows, cAMP levels, and cAMP-dependent protein kinase activity. Both water flow and activation of cAMP-kinase in response to vasopressin were enhanced after prostaglandin inhibition, consistent with inhibition of vasopressin-induced cAMP generation by endogenous prostaglandins. On the other hand exogeneous PGE stimulated cAMP generation. PGE1 (10(-7) M) alone did not increase water flow but activated kinase more than vasopressin only. Addition of PGE1 (10(-7) M) and vasopressin inhibited water flow as compared with vasopressin along but increased the kinase ratio above that with vasopressin only. PGE2 (10(-5) M) increased the cAMP content and kinase ratio even more than vasopressin but again resulted in no water flow. Addition of vasopressin and PGE2 (10(-5) M) increased water flow but did not alter cAMP content or the kinase ratio compared with PGE2 alone. Similar results were obtained with PGE1. Accordingly, prostaglandin dissociates cAMP levels and kinase ratio from the hydroosmotic response, suggesting that PGE2 inhibits steps distal to cAMP. Consistent with this, in bladders pretreated with naproxen or meclofenamate, PGE2 (10(-8) to 10(-6) M) inhibited the response to submaximal doses of cAMP (5 mM) or 8-bromo-cAMP (0.03 mM). Furthermore, pretreatment with naproxen significantly enhanced the response to cAMP (5 mM). These studies provide evidence for vasopressin-PGE interaction at the site of cAMP generation and also at a step(s) unrelated to cAMP generation.

Sign in / Sign up

Export Citation Format

Share Document