Hong-Jun Liao

Trafficking of receptor tyrosine kinases to the nucleus

It has been known for at least 20 years that growth factors induce the internalization of cognate receptor tyrosine kinases (RTKs). The internalized receptors are then sorted to lysosomes or recycled to the cell surface. More recently, data have been published to indicate other intracellular destinations for the internalized RTKs. These include the nucleus, mitochondria, and cytoplasm. Also, it is recognized that trafficking to these novel destinations involves new biochemical mechanisms, such as proteolytic processing or interaction with translocons, and that these trafficking events have a function in signal transduction, implicating the receptor itself as a signaling element between the cell surface and the nucleus.

Cetuximab/C225-Induced Intracellular Trafficking of Epidermal Growth Factor Receptor

The monoclonal antibody C225 interacts with the ectodomain of the epidermal growth factor (EGF) receptor (EGFR) to block ligand binding and initiates receptor endocytosis and intracellular trafficking. The data herein show that C225-dependent EGFR trafficking relocalizes the receptor to the endoplasmic reticulum (ER) and nucleus. This mechanism, which also involves interaction of the C225-internalized receptor with the Sec61 translocon within the ER, is, in most respects, analogous to the pathway previously described for EGF-induced trafficking to the ER and nucleus. However, although inhibition of receptor tyrosine kinase activity blocks EGF-induced nuclear localization of the receptor, the same kinase inhibitors stimulate C225-dependent nuclear localization of EGFR in the nucleus. In contrast, the kinase inhibitor Lapatinib fails to stimulate nuclear accumulation of the receptor in C225-treated cells and does not provoke receptor dimerization as do inhibitors that recognize the open conformation of the receptor kinase. This suggests that inhibitor-dependent receptor dimerization may facilitate C225-induced receptor trafficking.

Receptor Tyrosine Kinases in the Nucleus

To date, 18 distinct receptor tyrosine kinases (RTKs) are reported to be trafficked from the cell surface to the nucleus in response to ligand binding or heterologous agonist exposure. In most cases, an intracellular domain (ICD) fragment of the receptor is generated at the cell surface and translocated to the nucleus, whereas for a few others the intact receptor is translocated to the nucleus. ICD fragments are generated by several mechanisms, including proteolysis, internal translation initiation, and messenger RNA (mRNA) splicing. The most prevalent mechanism is intramembrane cleavage by γ-secretase. In some cases, more than one mechanism has been reported for the nuclear localization of a specific RTK. The generation and use of RTK ICD fragments to directly communicate with the nucleus and influence gene expression parallels the production of ICD fragments by a number of non-RTK cell-surface molecules that also influence cell proliferation. This review will be focused on the individual RTKs and to a lesser extent on other growth-related cell-surface transmembrane proteins.

Regulated Intramembrane Cleavage of the EGF Receptor

Following the addition of EGF or ionomycin to A431 cells, protease activity mediates cleavage of the EGF receptor producing a 60 kDa fragment that includes the intracellular domain (ICD). This fragment is located in both membrane and nuclear fractions. On the basis of sensitivity to chemical inhibitors and overexpression of cDNAs, the rhomboid intramembrane proteases, not γ‐secretase proteases, are identified as responsible for the cleavage event. Agonist‐initiated cleavage occurs slowly over 3–24 h. Inhibition of calpain protease activity significantly increased the detectable level of ICD fragment.

Phospholipase C-γ1 Is Required for the Induction of Immediate Early Genes by Platelet-derived Growth Factor

To explore the functional role of phospholipase C-γ1 (PLC-γ1) in the induction of immediate early genes (IEGs), we have examined the influence of Plcg1 gene disruption on the expression of 14 IEG mRNAs induced by platelet-derived growth factor (PDGF). Plcg1-null embryos were used to produce immortalized fibroblasts genetically deficient in PLC-γ1 (Null cells), and retroviral infection of those cells was used to derive PLC-γ1 re-expressing cells (Null+ cells). In terms of PDGF activation of PDGF receptor tyrosine phosphorylation as well as the mitogen-activated protein kinases Erk1 and Erk2, Null and Null+ cells responded equivalently. However, the PDGF-dependent expression of all IEG mRNAs was diminished in cells lacking PLC-γ1. The expression of FIC, COX-2, KC,JE, and c-fos mRNAs were most strongly compromised, as the stimulation of these genes was reduced by more than 90% in cells lacking PLC-γ1. The combination of PMA and ionomycin, downstream analogs of PLC activation, did provoke expression of mRNAs for these IEGs in the Null cells. We conclude that PLC-γ1 is necessary for the maximal expression of many PDGF-induced IEGs and is essential for significant induction of at least five IEGs.

Chapter 110 – Phospholipase C

Publisher Summary This chapter focuses on the phosphoinositide-specific phospholipase C (PLC) isozymes expressed in mammalian cells. It discusses molecular structure/function and activation mechanisms for phospholipase C enzymes and presents the physiologic consequences of PLC genetic knockouts. This enzyme uniquely activates two second messengers, which in turn may control a variety of signaling pathways and thereby influence a panoply of cellular events. Each PLC subgroup is characterized by additional motifs that are involved in regulating aspects of enzyme function, such as topological localization within the cell and sensitivity to protein–protein and protein–lipid interactions. Isozymes yield significant information regarding the molecular mechanisms by which these enzymes are activated in cells; there is much less information available regarding the role of these PLC isozymes in physiologic or pathophysiologic processes. The results, in some cases, represent phenotypes obtained at the first crucial point in development when a particular PLC isoform becomes required for further development of the organism.

Nuclear ErbB Receptors: Pathways and Functions

Accumulating evidence suggests a new mode of ErbB receptor signaling in which intact or fragmented ErbB receptors traffic from the cell surface to the nucleus following the addition of a cognate ligand. In the nucleus, ErbB receptors have a role, probably indirect, in modulating gene expression. Following the addition of growth factor, ErbB1 is sorted from the cell surface to the endoplasmic reticulum where it interacts with the Sec61 translocon and is retrotranslocated from the endoplasmic reticulum to cytoplasm. This is a precursor step for subsequent nuclear localization of the receptor and the induction of cyclin D by EGF. In the case of ErbB4, the receptor is processed by two membrane-localized proteases to produce a soluble cytoplasmic domain fragment that translocates to the nucleus. Nuclear ErbB1 and ErbB4 have been detected in the tissue of cancer patients and may portend a poorer prognosis. Less understood are mechanisms that provoke nuclear localization of ErbB2 and ErbB3, which have been described in cell culture systems.

CHAPTER 122 – Phospholipase C

This chapter focuses on the phosphoinositide-specific phospholipase C (PLC) isozymes expressed in mammalian cells. This family of isozymes is defined on the basis of sequence similarities and the capacity to mediate the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PI 4,5-P 2 ) to the second messenger molecules inositol 1,4,5-trisphosphate and diacylglycerol. The phospholipase C enzymes that hydrolyze phosphatidylinositol 4,5-bisphosphate in mammalian cells are subdivided into four families, denoted β, γ, δ, and ɛ, based on sequence similarities. Each family has a unique organization of regulatory sequence motifs or domains that facilitate protein: protein and/or protein: phospholipid interactions. Utilizing these motifs, each family responds to distinct hormonal signals or intracellular cues to produce the second messenger molecules inositol 1,4,5-trisphosphate and diacyl-glycerol. These metabolites in turn control intracellular levels of free Ca 2+ and protein kinase C activity, respectively. This chapter, in addition to discussing molecular structure/function and activation mechanisms for phospholipase C enzymes, presents the physiologic consequences of PLC genetic knockouts.