Travis J. Maures

SH2B1 (SH2-B) and JAK2: a multifunctional adaptor protein and kinase made for each other

Src homology 2 (SH2) B adaptor protein 1 (SH2B1; originally named SH2-B) is a member of a family of adaptor proteins that influences a variety of signaling pathways mediated by Janus kinase (JAK) and receptor tyrosine kinases. Although SH2B1 performs classical adaptor functions, such as recruitment of specific proteins to activated receptors, it also demonstrates a unique ability to enhance the kinase activity of the cytokine receptor-associated tyrosine kinase JAK2, as well as that of several receptor tyrosine kinases. SH2B1 is also among a small number of adaptor proteins shown to undergo nucleocytoplasmic shuttling, although its exact role within the nucleus is not yet clear. Deletion of the SH2B1 gene results in severe obesity and both leptin and insulin resistance, as well as infertility, which might be a consequence of resistance to insulin-like growth factor I. Thus, knockout mice support a role for SH2B1 as a positive regulator of JAK2 signaling pathways initiated by leptin, as well as of pathways initiated by insulin and, potentially, by insulin-like growth factor I.

Evidence That IGF Binding Protein-5 Functions as a Ligand-Independent Transcriptional Regulator in Vascular Smooth Muscle Cells

Abstract— Insulin-like growth factor binding protein (IGFBP)-5 is a conserved protein synthesized and secreted by vascular smooth muscle cells (VSMCs). IGFBP-5 binds to extracellular IGFs and modulates IGF actions in regulating VSMC proliferation, migration, and survival. IGFBP-5 also stimulates VSMC migration through an IGF-independent mechanism, but the molecular basis underlying this ligand-independent action is unknown. In this study, we show that endogenous IGFBP-5 or transiently expressed IGFBP-5-EGFP, but not IGFBP-4-EGFP, is localized in the nuclei of VSMCs. Using a series of IGFBP-4/5 chimeras and IGFBP-5 points mutants, we demonstrated that the IGFBP-5 C-domain is necessary and sufficient for its nuclear localization, and residues K206, K208, K217, and K218 are particularly critical. Intriguingly, inhibition of protein secretion abolishes IGFBP-5 nuclear localization, suggesting the nuclear IGFBP-5 is derived from the secreted protein. When added exogenously, 125I- or Cy3-labeled IGFBP-5 is capable of cellular entry and nuclear translocation. To identify potential transcriptional factor(s) that interact with IGFBP-5, a human aorta cDNA library was screened by a yeast two-hybrid screening strategy. Although this screen identified many extracellular and cytosolic proteins that are known to interact with IGFBP-5, no known transcription factors were found. Further motif analysis revealed that the IGFBP-5 N-domain contains a putative transactivation domain. When fused to GAL-4 DNA dinging domain and tested, the IGFBP-5 N-domain has strong transactivation activity. Mutation of the IGF binding domain or treatment of cells with IGF-I has little effect on transactivation activity. These results suggest that IGFBP-5 is localized in VSMC nucleus and possesses transcription-regulatory activity that is IGF independent. The full text of this article is available online at http://circres.ahajournals.org.

Ontogeny, tissue distribution, and hormonal regulation of insulin-like growth factor binding protein-2 (IGFBP-2) in a marine fish, Sparus aurata

In this study, we have cloned insulin-like growth factor binding protein (IGFBP)-2 from a marine hermaphroditic fish species, the gilthead sea bream (Sparus aurata), and determined its structure, ontogeny, tissue distribution, and hormonal regulation. The sea bream IGFBP-2 precursor consists of 286 amino acids, including a putative signal peptide of 22 residues and a mature protein of 264 residues. The overall sequence of sea bream preIGFBP-2 is 52-39% identical to that of zebrafish, chick, mouse, rat, pig, sheep, bovine, and human preIGFBP-2. The cysteine-rich N- and C-terminal domains, which are believed to be important for IGF binding, show a greater degree of conservation with a sequence identity of 73-56% and 53-50%, respectively. Sea bream IGFBP-2 contains 18 cysteine residues and their alignment is identical to those of other vertebrate IGFBP-2s. The Arg-Gly-Asp (RGD) sequence, present in the C-terminal domain of all known IGFBP-2, is also present in the sea bream. Northern blot analysis of RNA samples extracted from adult liver and developing larvae, revealed two transcripts of about 1.3 and 2.5 kb. RT-PCR analysis showed that IGFBP-2 was expressed in all adult tissues studied, with the highest levels found in liver and skin. IGFBP-2 transcripts were detected in gonad during reproductive cycle of S. aurata. Highest levels of IGFBP-2 mRNA were found in bisexual young gonads, and the levels decreased with gonad development and relatively high levels of IGFBP-2 mRNA were found in the ovary during spawning. By contrast, testicular IGFBP-2 mRNA levels were very low. RT-PCR detected IGFBP-2 mRNA throughout development in unfertilized eggs, embryos, and larvae, with highest levels observed after day 3 post-hatching, suggesting that this mRNA is the product of both the maternal and embryonic genomes. High variability in steady-state levels of hepatic IGFBP-2 mRNA was noted in adult fish, resulting in a statistically insignificant response to growth hormone treatment. These results suggest that the structure of IGFBP-2 is conserved in sea bream and that IGFBP-2 mRNA is expressed during early development and in gonad during the reproductive cycle, suggesting that it may play a role in gilthead sea bream development and reproduction.

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.

Nucleocytoplasmic Shuttling of the Adapter Protein SH2B1β (SH2-Bβ) Is Required for Nerve Growth Factor (NGF)-Dependent Neurite Outgrowth and Enhancement of Expression of a Subset of NGF-Responsive Genes

The adapter protein SH2B1 (SH2-B, PSM) is recruited to multiple ligand-activated receptor tyrosine kinases, including the receptors for nerve growth factor (NGF), insulin, and IGF-I as well as the cytokine receptor-associated Janus kinase family kinases. In this study, we examine SH2B1s function in NGF signaling. We show that depleting endogenous SH2B1 using short hairpin RNA against SH2B1 inhibits NGF-dependent neurite outgrowth, but not NGF-mediated phosphorylation of Akt or ERKs 1/2. SH2B1 has been hypothesized to localize and function at the plasma membrane. We identify a nuclear localization signal within SH2B1 and show that it is required for nuclear translocation of SH2B1beta. Mutation of the nuclear localization signal has no effect on NGF-induced activation of TrkA and ERKs 1/2 but prevents SH2B1beta from enhancing NGF-induced neurite outgrowth. Disruption of SH2B1beta nuclear import also prevents SH2B1beta from enhancing NGF-induced transcription of genes important for neuronal differentiation, including those encoding urokinase plasminogen activator receptor, and matrix metalloproteinases 3 and 10. Disruption of SH2B1beta nuclear export by mutation of its nuclear export sequence similarly prevents SH2B1beta enhancement of NGF-induced transcription of those genes. Nuclear translocation of the highly homologous family member SH2B2(APS) was not observed. Together, these data suggest that rather than simply acting as an adapter protein linking signaling proteins to the activated TrkA receptor at the plasma membrane, SH2B1beta must shuttle between the plasma membrane and nucleus to function as a critical component of NGF-induced gene expression and neuronal differentiation.

Phosphorylation controls a dual-function polybasic nuclear localization sequence in the adapter protein SH2B1β to regulate its cellular function and distribution

An intriguing question in cell biology is what targets proteins to, and regulates their translocation between, specific cellular locations. Here we report that the polybasic nuclear localization sequence (NLS) required for nuclear entry of the adapter protein and candidate human obesity gene product SH2B1β, also localizes SH2B1β to the plasma membrane (PM), most probably via electrostatic interactions. Binding of SH2B1β to the PM also requires its dimerization domain. Phosphorylation of serine residues near this polybasic region, potentially by protein kinase C, releases SH2B1β from the PM and enhances nuclear entry. Release of SH2B1β from the PM and/or nuclear entry appear to be required for SH2B1β enhancement of nerve growth factor (NGF)-induced expression of urokinase plasminogen activator receptor gene and neurite outgrowth of PC12 cells. Taken together, our results provide strong evidence that the polybasic NLS region of SH2B1 serves the dual function of localizing SH2B1 to both the nucleus and the PM, the latter most probably through electrostatic interactions that are enhanced by SH2B1β dimerization. Cycling between the different cellular compartments is a consequence of the phosphorylation and dephosphorylation of serine residues near the NLS and is important for physiological effects of SH2B1, including NGF-induced gene expression and neurite outgrowth.

Identification of Steroid-Sensitive Gene-1/Ccdc80 as a JAK2-Binding Protein

The tyrosine kinase Janus kinase 2 (JAK2) is activated by many cytokine receptors, including receptors for GH, leptin, and erythropoietin. However, very few proteins have been identified as binding partners for JAK2. Using a yeast 2-hybrid screen, we identified steroid-sensitive gene-1 (SSG1)/coiled-coil domain-containing protein 80 (Ccdc80) as a JAK2-binding partner. We demonstrate that Ccdc80 preferentially binds activated, tyrosyl-phosphorylated JAK2 but not kinase-inactive JAK2 (K882E) in both yeast and mammalian systems. Ccdc80 is tyrosyl phosphorylated in the presence of JAK2. The binding of Ccdc80 to JAK2 occurs via 1 or more of the 3 DUDES/SRPX (DRO1-URB-DRS-Equarin-SRPUL/sushi repeat containing protein, x-linked) domain 5 domains of Ccdc80. Mutagenesis of the second DUDES domain suggests that the N-terminal third of the DUDES domain is sufficient for JAK2 binding. Ccdc80 does not alter the kinase activity of JAK2. However, Ccdc80 increases GH-dependent phosphorylation of Stat (signal transducer and activator of transcription) 5b on Tyr699 and substantially enhances both basal and GH-dependent phosphorylation/activation of Stat3 on Tyr705. Furthermore, Ccdc80 belongs to the group of proteins that function both in the intracellular compartment and are secreted. Secreted Ccdc80 associates with the extracellular matrix and is also found in the medium. A substantial portion of the Ccdc80 detected in the medium is cleaved. Finally, consistent with the DUDES domain serving as a JAK2-binding domain, we also demonstrate that another protein that contains a DUDES domain, SRPX2, binds preferentially to the activated tyrosyl-phosphorylated form of JAK2.