ABSTRACT
Conditioned stimulus pathway protein 24 (Csp24) is a beta-thymosin-like protein that is homologous to other members of the family of beta-thymosin repeat proteins that contain multiple actin binding domains. Actin co-precipitates with Csp24 and co-localizes with it in the cytosol of type-B photoreceptor cell bodies. Several signal transduction pathways have been shown to regulate the phosphorylation of Csp24 and contribute to cellular plasticity. Here, we report the identification of the adapter protein 14-3-3 in lysates of the Hermissenda circumesophageal nervous system and its interaction with Csp24. Immunoprecipitation experiments using an antibody that is broadly reactive with several isoforms of the 14-3-3 family of proteins showed that Csp24 co-precipitates with 14-3-3 protein, and nervous systems stimulated with 5-HT exhibited a significant increase in co-precipitated Csp24 probed with a phosphospecific antibody as compared with controls. These results indicate that post-translational modifications of Csp24 regulate its interaction with 14-3-3 protein, and suggest that this mechanism may contribute to the control of intrinsic enhanced excitability.
Subject(s)
14-3-3 Proteins/metabolism , Ganglia, Invertebrate/metabolism , Hermissenda/metabolism , Microfilament Proteins/metabolism , Nervous System/metabolism , Phosphoproteins/metabolism , Animals , Ganglia, Invertebrate/drug effects , Immunohistochemistry , MAP Kinase Signaling System/drug effects , MAP Kinase Signaling System/physiology , Phosphorylation/drug effects , Photoreceptor Cells, Invertebrate/metabolism , Protein Processing, Post-Translational/physiology , Serotonin/metabolism , Serotonin/pharmacology , Signal Transduction/drug effects , Signal Transduction/physiology , Thymosin/analogs & derivatives , Thymosin/metabolism , Ubiquitins/metabolismABSTRACT
The regulation of the intrinsic excitability of a neuron is an important aspect of cellular and synaptic plasticity underlying learning and memory. Various voltage-dependent K(+) channels have been shown to be critical for the modification of membrane excitability. Components of the cytoskeleton have been proposed to contribute to the location, distribution, and function of diverse K(+) channels. However, the mechanisms underlying the regulation of the cytoskeleton by signaling pathways and the role of the cytoskeleton in the induction of intrinsic excitability is not understood. Hermissenda Csp24 is a beta-thymosin-like protein containing multiple actin-binding domains that contributes to intrinsic enhanced excitability produced by Pavlovian conditioning. One-trial in vitro conditioning produces a significant reduction in the A-type transient K(+) current (I(A)) and a depolarized shift in the steady-state activation curve of I(A). Intermediate and long-term enhanced excitability produced by one-trial conditioning is also dependent on the expression and phosphorylation of Csp24. Blocking the expression of Csp24 with an antisense oligonucleotide inhibits the development of intermediate-term enhanced excitability and the concomitant reduction in I(A) normally produced by one-trial in vitro conditioning. In this report using two-dimensional gel PAGE and electrospray mass spectrometry, we have identified two phosphorylation sites on Csp24. Using phospho-specific antibodies with Western blot analysis and immunoprecipitation procedures we show that one-trial in vitro conditioning results in an increase in the phosphorylation of Ser-122, but not Ser-49 of Csp24.
Subject(s)
Hermissenda/physiology , Microfilament Proteins/metabolism , Phosphoproteins/metabolism , Phosphoserine/metabolism , Amino Acid Sequence , Animals , Echocardiography , Microfilament Proteins/chemistry , Microfilament Proteins/isolation & purification , Molecular Sequence Data , Nerve Tissue Proteins/metabolism , Nervous System Physiological Phenomena , Phosphoproteins/chemistry , Phosphoproteins/isolation & purification , Phosphorylation , Potassium/physiology , Thymosin/physiologyABSTRACT
Rho GTPases acting through effector proteins regulate actin dynamics and cytoskeletal structure. In Hermissenda Csp24 is a cytoskeletal-related protein that contributes to the development of intermediate-term memory, and is homologous to other beta-thymosin-like repeat proteins containing multiple actin-binding domains. We have examined the role of Rho GTPase activity and its downstream target ROCK, and cyclin-dependent kinase 5 (Cdk5) on the phosphorylation of Csp24 using 32PO4 labeling of proteins separated with 2-D PAGE. The ROCK inhibitor Y-27632 significantly increased Csp24 phosphorylation, and the Rho activator lysophosphatidic acid (LPA) or the Cdk5 inhibitor butyrolactone significantly decreased Csp24 phosphorylation. Pretreatment with Y-27632 before LPA application significantly reduced the decreased phosphorylation of Csp24 normally detected in nervous systems exposed to LPA. Using a pull-down assay we found that LPA treatments activated Rho and exposure to 5-HT decreased Rho activity. Our results indicate that the Rho/ROCK and Cdk5 signaling pathways contribute to the regulation of Csp24 phosphorylation.
