James Herrington

Signaling pathways activated by the growth hormone receptor

In recent years, significant progress has been made in elucidating the signaling pathways activated by the growth hormone (GH) receptor. An initiating event is probably the activation of JAK2 (Janus kinase 2), a GH receptor-associated tyrosine kinase. Identification of the proteins recruited to the GH receptor-JAK2 complex and dissection of the signaling pathways that are subsequently activated will ultimately provide a basis for understanding GH action at the molecular level.

SH2-B Is Required for Nerve Growth Factor-induced Neuronal Differentiation

Nerve growth factor (NGF) is essential for the development and survival of sympathetic and sensory neurons. NGF binds to TrkA, activates the intrinsic kinase activity of TrkA, and promotes the differentiation of pheochromocytoma (PC12) cells into sympathetic-like neurons. Several signaling molecules and pathways are known to be activated by NGF, including phospholipase Cγ, phosphatidylinositol-3 kinase, and the mitogen-activated protein kinase cascade. However, the mechanism of NGF-induced neuronal differentiation remains unclear. In this study, we examined whether SH2-Bβ, a recently identified pleckstrin homology and SH2 domain-containing signaling protein, is a critical signaling protein for NGF. TrkA bound to glutathione S-transferase fusion proteins containing SH2-Bβ, and NGF stimulation dramatically increased that binding. In contrast, NGF was unable to stimulate the association of TrkA with a glutathione S-transferase fusion protein containing a mutant SH2-Bβ(R555E) with a defective SH2 domain. When overexpressed in PC12 cells, SH2-Bβ co-immunoprecipitated with TrkA in response to NGF. NGF stimulated tyrosyl phosphorylation of endogenous SH2-Bβ as well as exogenously expressed GFP-SH2-Bβ but not GFP-SH2-Bβ(R555E). Overexpression of SH2-Bβ(R555E) blocked NGF-induced neurite outgrowth of PC12 cells, whereas overexpression of wild type SH2-Bβ enhanced NGF-induced neurite outgrowth. Overexpression of either wild type or mutant SH2-Bβ(R555E) did not alter tyrosyl phosphorylation of TrkA, Shc, or phospholipase Cγ in response to NGF or NGF-induced activation of ERK1/2, suggesting that SH2-Bβ may initiate a previously unknown pathway(s) that is essential for NGF-induced neurite outgrowth. Taken together, these data indicate that SH2-Bβ is a novel signaling molecule required for NGF-induced neuronal differentiation.

Differential Binding to and Regulation of JAK2 by the SH2 Domain and N-Terminal Region of SH2-Bβ

ABSTRACT SH2-Bβ has been shown to bind via its SH2 (Src homology 2) domain to tyrosyl-phosphorylated JAK2 and strongly activate JAK2. In this study, we demonstrate the existence of an additional binding site(s) for JAK2 within the N-terminal region of SH2-Bβ (amino acids 1 to 555) and the ability of this region of SH2-B to inhibit JAK2. Four lines of evidence support the existence of this additional binding site(s). In a glutathione S-transferase pull-down assay, wild-type SH2-Bβ and SH2-Bβ(R555E) with a defective SH2 domain bind to both tyrosyl-phosphorylated JAK2 from growth hormone (GH)-treated cells and non-tyrosyl-phosphorylated JAK2 from control cells, whereas the SH2 domain of SH2-Bβ binds only to tyrosyl-phosphorylated JAK2 from GH-treated cells. Similarly, JAK2 is present in αSH2-B immunoprecipitates in the absence and presence of GH, with GH substantially increasing the coprecipitation of JAK2 with SH2-B. When coexpressed in COS cells, SH2-Bβ coimmunoprecipitates not only wild-type, tyrosyl-phosphorylated JAK2 but also kinase-inactive, non-tyrosyl-phosphorylated JAK2(K882E), although to a lesser extent. ΔC555 (amino acids 1 to 555 of SH2-Bβ) that lacks most of the SH2 domain binds similarly to wild-type JAK2 and kinase-inactive JAK2(K882E). Experiments using a series of N- and C-terminally truncated SH2-Bβ constructs indicate that the pleckstrin homology (PH) domain (amino acids 269 to 410) and amino acids 410 to 555 are necessary for maximal binding of SH2-Bβ to inactive JAK2, but neither region alone is sufficient for maximal binding. The SH2 domain of SH2-Bβ is necessary and sufficient for the stimulatory effect of SH2-Bβ on JAK2 and JAK2-mediated tyrosyl phosphorylation of Stat5B. In contrast, ΔC555 lacking the SH2 domain, and to a lesser extent the PH domain alone, inhibits JAK2. ΔC555 also blocks JAK2-mediated tyrosyl phosphorylation of Stat5B in COS cells and GH-stimulated nuclear accumulation of Stat5B in 3T3-F442A cells. These data indicate that in addition to the SH2 domain, SH2-Bβ has one or more lower-affinity binding sites for JAK2 within amino acids 269 to 555. The interaction via this site(s) in SH2-B with inactive JAK2 seems likely to increase the local concentration of SH2-Bβ around JAK2, thereby facilitating binding of the SH2 domain to ligand-activated JAK2. This would result in a more rapid and robust cellular response to hormones and cytokines that activate JAK2. This interaction between inactive JAK2 and SH2-B may also help prevent abnormal activation of JAK2.

SH2-Bβ is a Rac binding protein that regulates cell motility

The Src homology 2 (SH2) domain-containing protein SH2-Bβ binds to and is a substrate of the growth hormone (GH) and cytokine receptor-associated tyrosine kinase JAK2. SH2-Bβ also binds, via its SH2 domain, to multiple activated growth factor receptor tyrosine kinases. We have previously implicated SH2-Bβ in GH and platelet-derived growth factor regulation of the actin cytoskeleton. We extend these findings by establishing a potentiating effect of SH2-Bβ on GH-dependent cell motility and defining regions of SH2-Bβ required for this potentiation. Time-lapse video microscopy, phagokinetic, and/or wounding assays demonstrate reduced movement of cells overexpressing SH2-Bβ lacking an intact SH2 domain because of a point mutation or a C-terminal truncation. An N-terminal proline-rich domain (amino acids 85–106) of SH2-Bβ is required for inhibition of cellular motility by SH2 domain-deficient mutants. Co-immunoprecipitation experiments indicate that Rac binds to this domain. GH is shown to activate endogenous Rac, and dominant negative mutants of SH2-Bβ are shown to inhibit membrane ruffling induced by constitutively active Rac. These findings suggest that SH2-Bβ is an adapter protein that facilitates actin rearrangement and cellular motility by recruiting Rac and potentially Rac-regulating, Rac effector, or other actin-regulating proteins to activated cytokine (e.g. GH) and growth factor receptors.

Role of the tyrosine kinase JAK2 in signal transduction by growth hormone

Abstract Chronic renal failure in children results in impaired body growth. This effect is so severe in some children that not only does it have a negative impact on their self-image, but it also affects their ability to carry out normal day-to-day functions. Yet the mechanism by which chronic renal failure causes short stature is not well understood. Growth hormone (GH) therapy increases body height in prepubertal children, suggesting that a better understanding of how GH promotes body growth may lead to better insight into the impaired body growth in chronic renal failure and therefore better therapies. This review discusses what is currently known about how GH acts at a cellular level. The review discusses how GH is known to bind to a membrane-bound receptor and activate a cytoplasmic tyrosine kinase called Janus kinase (JAK) 2. The activated JAK2 in turn phosphorylates tyrosines within itself and the associated GH receptor, forming high-affinity binding sites for a variety of signaling molecules. Examples of such signaling molecules include signal transducers and activators of transcription (Stats), which regulate the expression of a variety of GH-dependent genes, and the adapter protein Shc, which leads to activation of the Ras-Raf-MEK-MAP kinase pathway. In response to GH, JAK2 is also known to phosphorylate the insulin receptor substrates, leading to activation of phosphatidyl inositol 3’ kinase and most likely other molecules that have been implicated in the regulation of metabolism. Finally, the ability of JAK2 to bind and activate the presumed adapter protein SH2-B is discussed. SH2-B has been shown to be a potent activator of GH-promoted JAK2 activity and downstream signaling events. Presumably these and other pathways initiated by GH combine to result in its ability to regulate body growth and metabolism.

SH2-B, a Membrane-associated Adapter, Is Phosphorylated on Multiple Serines/Threonines in Response to Nerve Growth Factor by Kinases within the MEK/ERK Cascade

SH2-B has been shown to be required for nerve growth factor (NGF)-mediated neuronal differentiation and survival, associate with NGF receptor TrkA, and be tyrosyl-phosphorylated in response to NGF. In this work, we examined whether NGF stimulates phosphorylation of SH2-B on serines/threonines. NGF promotes a dramatic upward shift in mobility of SH2-B, resulting in multiple forms that cannot be attributed to tyrosyl phosphorylation. Treatment of SH2-B with protein phosphatase 2A, a serine/threonine phosphatase, reduces the many forms to two. PD98059, a MEK inhibitor, dramatically inhibits NGF-promoted phosphorylation of SH2-B on serines/threonines, whereas depletion of 4β-phorbol 12-myristate 13-acetate-sensitive protein kinase Cs does not. ERKs 1 and 2 phosphorylate SH2-Bβ primarily on Ser-96 in vitro. However, NGF still stimulates serine/threonine phosphorylation of SH2-Bβ(S96A). SH2-Bβ(S96A), like wild-type SH2-Bβ, enhances NGF-induced neurite outgrowth. In contrast, SH2-Bβ(R555E) containing a defective SH2 domain blocks NGF-induced neurite outgrowth and displays greatly reduced phosphorylation on serines/threonines in response to NGF. SH2-Bβ(R555E), like wild-type SH2-Bβ, associates with the plasma membrane, suggesting that the dominant negative effect of SH2-Bβ(R555E) cannot be explained by an abnormal subcellular distribution. In summary, NGF stimulates phosphorylation of SH2-B on serines/threonines by kinases downstream of MEK, which may be important for NGF-mediated neuronal differentiation and survival.

SH2-B Is a Positive Regulator of Nerve Growth Factor-mediated Activation of the Akt/Forkhead Pathway in PC12 Cells

To gain insight into the mechanism by which the adapter protein SH2-B promotes nerve growth factor (NGF)-mediated neuronal differentiation and survival, the effect of SH2-B on the serine/threonine kinase Akt/protein kinase B and downstream effector proteins was examined. PC12 cells stably overexpressing SH2-Bβ, which exhibit enhanced NGF-induced neuronal differentiation compared with control cells, showed enhanced and prolonged NGF-induced phosphorylation of Akt on Ser473 and Akt enzymatic activity. Surprisingly, NGF-induced phosphorylation of Akt on Ser473 and Akt activity were not altered in cells overexpressing SH2-Bβ(R555E) with a defective SH2 domain, despite the ability of the overexpressed SH2-Bβ(R555E) to block NGF-induced differentiation. Consistent with SH2-Bβ enhancing the activity of Akt, cells overexpressing SH2-Bβ but not SH2-Bβ(R555E) exhibited increased and/or prolonged phosphorylation of the pro-apoptotic Akt effector proteins, glycogen synthase kinase-3, and forkhead transcription factors, FKHRL1/FOXO3 and FKHR/FOXO1. Immunolocalization studies indicated that, although ectopically expressed FKHR was primarily concentrated in the cytoplasm of control cells and cells transiently overexpressing SH2-Bβ, it was concentrated in the nucleus of cells transiently overexpressing SH2-Bβ(R555E). Similarly, SH2-Bβ stimulated the accumulation of FKHR in the cytoplasm of 293T and COS-7 cells, whereas SH2-Bβ(R555E) enhanced its accumulation in the nucleus. In PC12 cells stably expressing forms of SH2-Bβ, SH2-Bβ mimicked the ability of NGF to promote redistribution of FKHR to the cytoplasm whereas SH2-Bβ(R555E) blocked this effect of NGF. Taken together, these data indicate that SH2-B is a positive regulator of NGF-mediated activation of the Akt/Forkhead pathway.