ABSTRACT
Current therapeutical approaches for the treatment of severe lesions in the peripheral nervous system rely on the use of autologous tissue or the body's own Schwann cells. However, these approaches are limited and alternative strategies for peripheral nerve regeneration are required. Here we evaluate combinations of a variety of neuronal regeneration factors including engineered cell adhesion molecules and growth factors in embryonic model neurons to test the possible improvement of artificial nerve guides by cooperative mechanisms. Cell adhesion molecules L1 and neurofascin synergistically promote neurite elongation. The outgrowth promoting properties of both proteins can be combined and further increased within one chimeric protein. Addition of growth and differentiation factor 5 (GDF-5) further enhances neurite outgrowth in a substrate-independent manner. This effect is not due to a protective mode of action of GDF-5 against pro-apoptotic stimuli. Consequently, the study supports the idea that different modes of action of pro-regenerative factors may contribute synergistically to neurite outgrowth and emphasizes the applicability of combinations of proteins specifically involved in development of the nervous system for therapeutical approaches.
Subject(s)
Bone Morphogenetic Proteins/pharmacology , Nerve Regeneration/drug effects , Neural Cell Adhesion Molecules/pharmacology , Neurites/drug effects , Animals , Apoptosis , Chick Embryo , Growth Differentiation Factor 5 , Humans , Neurites/physiology , Neurons/drug effects , Neurons/physiology , Protein Engineering , Recombinant Fusion Proteins/pharmacologyABSTRACT
Protein kinase D2 (PKD2) belongs to the PKD family of serine/threonine kinases that is activated by phorbol esters and G protein-coupled receptors (GPCRs). Its C-terminal regulatory domain comprises two cysteine-rich domains (C1a/C1b) followed by a pleckstrin homology (PH) domain. Here, we examined the role of the regulatory domain in PKD2 phorbol ester binding, catalytic activity, and subcellular localization: The PH domain is a negative regulator of kinase activity. C1a/C1b, in particular C1b, is required for phorbol ester binding and gastrin-stimulated PKD2 activation, but it has no inhibitory effect on the catalytic activity. Gastrin triggers nuclear accumulation of PKD2 in living AGS-B cancer cells. C1a/C1b, not the PH domain, plays a complex role in the regulation of nucleocytoplasmic shuttling: We identified a nuclear localization sequence in the linker region between C1a and C1b and a nuclear export signal in the C1a domain. In conclusion, our results define the critical components of the PKD2 regulatory domain controlling phorbol ester binding, catalytic activity, and nucleocytoplasmic shuttling and reveal marked differences to the regulatory properties of this domain in PKD1. These findings could explain functional differences between PKD isoforms and point to a functional role of PKD2 in the nucleus upon activation by GPCRs.
Subject(s)
Nucleocytoplasmic Transport Proteins/chemistry , Nucleocytoplasmic Transport Proteins/physiology , Phorbol Esters/metabolism , Protein Kinases/chemistry , Protein Kinases/physiology , Catalysis , Cell Line , Cell Line, Tumor , Humans , Karyopherins/physiology , Nucleocytoplasmic Transport Proteins/genetics , Nucleocytoplasmic Transport Proteins/metabolism , Phorbol 12,13-Dibutyrate/metabolism , Protein Kinase C/chemistry , Protein Kinase C/genetics , Protein Kinase C/metabolism , Protein Kinase C/physiology , Protein Kinase D2 , Protein Kinases/genetics , Protein Kinases/metabolism , Protein Sorting Signals/physiology , Protein Structure, Tertiary , Protein Transport/physiology , Receptors, Cytoplasmic and Nuclear/physiology , Stomach Neoplasms/enzymology , Tritium , Exportin 1 ProteinABSTRACT
Recently, we cloned a novel serine/threonine kinase termed protein kinase D2 (PKD2). PKD2 can be activated by phorbol esters both in vivo and in vitro but also by gastrin via the cholecystokinin/CCK(B) receptor in human gastric cancer cells stably transfected with the CCK(B)/gastrin receptor (AGS-B cells). Here we identify the mechanisms of gastrin-induced PKD2 activation in AGS-B cells. PKD2 phosphorylation in response to gastrin was rapid, reaching a maximum after 10 min of incubation. Our data demonstrate that gastrin-stimulated PKD2 activation involves a heterotrimeric G alpha(q) protein as well as the activation of phospholipase C. Furthermore, we show that PKD2 can be activated by classical and novel members of the protein kinase C (PKC) family such as PKC alpha, PKC epsilon, and PKC eta. These PKCs are activated by gastrin in AGS-B cells. Thus, PKD2 is likely to be a novel downstream target of specific PKCs upon the stimulation of AGS-B cells with gastrin. Our data suggest a two-step mechanism of activation of PKD2 via endogenously produced diacylglycerol and the activation of PKCs.
Subject(s)
Protein Kinases/metabolism , Receptors, Cholecystokinin/metabolism , Cell Line , Enzyme Activation , Gastrins/metabolism , Heterotrimeric GTP-Binding Proteins/metabolism , Humans , Phosphorylation , Protein Kinase D2 , Signal Transduction , Type C Phospholipases/metabolismABSTRACT
Recent research has identified protein kinase D (PKD, also called PKCmu) as a serine/threonine kinase with potentially important roles in growth factor signaling as well as in stress-induced signaling. Moreover, PKD has emerged as an important regulator of plasma membrane enzymes and receptors, in some cases mediating cross-talk between different signaling systems. The recent discovery of two additional kinases belonging to the PKD family and the plethora of proteins that interact with PKD point to a multifaceted regulation and a multifunctional role for these enzymes, with functions in processes as diverse as cell proliferation, apoptosis, immune cell regulation, tumor cell invasion and regulation of Golgi vesicle fission.
