Xin-Yun Huang

Src Tyrosine Kinase Is a Novel Direct Effector of G Proteins

Heterotrimeric G proteins transduce signals from cell surface receptors to modulate the activity of cellular effectors. Src, the product of the first characterized proto-oncogene and the first identified protein tyrosine kinase, plays a critical role in the signal transduction of G protein-coupled receptors. However, the mechanism of biochemical regulation of Src by G proteins is not known. Here we demonstrate that Galphas and Galphai, but neither Galphaq, Galpha12 nor Gbetay, directly stimulate the kinase activity of downregulated c-Src. Galphas and Galphai similarly modulate Hck, another member of Src-family tyrosine kinases. Galphas and Galphai bind to the catalytic domain and change the conformation of Src, leading to increased accessibility of the active site to substrates. These data demonstrate that the Src family tyrosine kinases are direct effectors of G proteins.

Orai1 and STIM1 Are Critical for Breast Tumor Cell Migration and Metastasis

Tumor metastasis is the primary cause of death of cancer patients. Understanding the molecular mechanisms underlying tumor metastasis will provide potential drug targets. We report here that Orai1 and STIM1, both of which are involved in store-operated calcium entry, are essential for breast tumor cell migration in vitro and tumor metastasis in mice. Reduction of Orai1 or STIM1 by RNA interference in highly metastatic human breast cancer cells or treatment with a pharmacological inhibitor of store-operated calcium channels decreased tumor metastasis in animal models. Our data demonstrate a role for Orai1 and STIM1 in tumor metastasis and suggest store-operated calcium entry channels as potential cancer therapeutic targets.

Structural Basis for Relief of Autoinhibition of the Dbl Homology Domain of Proto-Oncogene Vav by Tyrosine Phosphorylation

Rho-family GTPases transduce signals from receptors leading to changes in cell shape and motility, mitogenesis, and development. Proteins containing the Dbl homology (DH) domain are responsible for activating Rho GTPases by catalyzing the exchange of GDP for GTP. Receptor-initiated stimulation of Dbl protein Vav exchange activity involves tyrosine phosphorylation. We show through structure determination that the mVav1 DH domain is autoinhibited by an N-terminal extension, which lies in the GTPase interaction site. This extension contains the Tyr174 Src-family kinase recognition site, and phosphorylation or truncation of this peptide results in stimulation of GEF activity. NMR spectroscopy data show that the N-terminal peptide is released from the DH domain and becomes unstructured upon phosphorylation. Thus, tyrosine phosphorylation relieves autoinhibition by exposing the GTPase interaction surface of the DH domain, which is obligatory for Vav activation.

Tyrosine kinase-dependent suppression of a potassium channel by the G protein-coupled m1 muscarinic acetylcholine receptor.

Neurotransmitter receptors alter membrane excitability and synaptic efficacy by generating intracellular signals that ultimately change the properties of ion channels. Through expression studies in Xenopus oocytes and mammalian cells, we found that the G protein-coupled m1 muscarinic acetylcholine receptor potently suppresses a cloned delayed rectifier K+ channel through a pathway involving phospholipase C activation and direct tyrosine phosphorylation of the K+ channel. Furthermore, analysis of neuroblastoma cells revealed that a similar tyrosine kinase-dependent pathway links endogenous G protein-coupled receptors to suppression of the native RAK channel. These results suggest a novel mechanism by which neurotransmitters and hormones may regulate a specific type of K+ channel that is widely expressed in the mammalian brain and heart.

Leucine-rich repeat kinase 2 (LRRK2)/PARK8 possesses GTPase activity that is altered in familial Parkinson’s disease R1441C/G mutants

Mutations in Leucine‐rich repeat kinase 2 (LRRK2) are linked to the most common familial forms and some sporadic forms of Parkinson’s disease (PD). The LRRK2 protein contains two well‐known functional domains, MAPKKK‐like kinase and Rab‐like GTPase domains. Emerging evidence shows that LRRK2 contains kinase activity which is enhanced in several PD‐associated mutants of LRRK2. However, the GTPase activity of LRRK2 has yet to be formally demonstrated. Here, we produced and purified the epitope‐tagged LRRK2 protein from transgenic mouse brain, and showed that purified brain LRRK2 possesses both kinase and GTPase activity as assayed by GTP binding and hydrolysis. The brain LRRK2 is associated with elevated kinase activity in comparison to that from transgenic lung or transfected cultured cells. In transfected cell cultures, we detected GTP hydrolysis activity in full‐length as well as in GTPase domain of LRRK2. This result indicates that LRRK2 GTPase can be active independent of LRRK2 kinase activity (while LRRK2 kinase activity requires the presence of LRRK2 GTPase as previously shown). We further found that PD mutation R1441C/G in the GTPase domain causes reduced GTP hydrolysis activity, consistent with the altered enzymatic activity in the mutant LRRK2 carrying PD familial mutations. Therefore, our study shows the biochemical characteristics of brain‐specific LRRK2 which is associated with robust kinase and GTPase activity. The distinctive levels of kinase/GTPase activity in brain LRRK2 may help explain LRRK2‐associated neuronal functions or dysfunctions in the pathogenesis of PD.

The G protein G-alpha-12 stimulates Bruton's tyrosine kinase and a rasGAP through a conserved PH/BM domain

Heterotrimeric guanine-nucleotide-binding proteins (G proteins) are signal transducers that relay messages from many receptors on the cell surface to modulate various cellular processes. The direct downstream effectors of G proteins consist of the signalling molecules that are activated by their physical interactions with a Gα or Gβγ subunit. Effectors that interact directly with Gα12 G proteins have yet to be identified,. Here we show that Gα12 binds directly to, and stimulates the activity of, Brutons tyrosine kinase (Btk) and a Ras GTPase-activating protein, Gap1m, in vitro and in vivo. Gα12 interacts with a conserved domain, composed of the pleckstrin-homology domain and the adjacent Btk motif, that is present in both Btk and Gap1m. Our results are, to our knowledge, the first to identify direct effectors for Gα12 and to show that there is a direct link between heterotrimeric and monomeric G proteins.

Direct stimulation of Bruton's tyrosine kinase by Gq-protein alpha-subunit

Heterotrimeric guanine-nucleotide-binding regulatory proteins (G proteins) transduce signals from a wide variety of cell-surface receptors to generate physiological responses. Protein-tyrosine kinases are another group of critical cellular signal transducers and their malfunction often leads to cancer. Although activation of G-protein-coupled receptors can elicit rapid stimulation of cellular protein-tyrosine phosphorylation, the mechanism used by G proteins to activate protein-tyrosine kinases is unclear. Here we show that the purified α-subunit of the Gq class of G proteins (Gαq) directly stimulates the activity of a purified non-receptor kinase, Brutons tyrosine kinase (Btk), whereas purified α-subunits from Gi1, GO or Gz proteins do not. Gαq can also activate Btk in vivo. Furthermore, in Btk-deficient cells, stimulation of another kinase, a p38 MAP kinase, by Gq-coupled receptors is blocked. Our results demonstrate that certain protein-tyrosine kinases can be direct effectors of G proteins.

Crystal structure of oligomeric β1-adrenergic G protein–coupled receptors in ligand-free basal state

G protein–coupled receptors (GPCRs) mediate transmembrane signaling. Before ligand binding, GPCRs exist in a basal state. Crystal structures of several GPCRs bound with antagonists or agonists have been solved. However, the crystal structure of the ligand-free basal state of a GPCR, the starting point of GPCR activation and function, had not yet been determined. Here we report the X-ray crystal structure of the ligand-free basal state of a GPCR in a lipid membrane–like environment. Oligomeric turkey β1-adrenergic receptors display two dimer interfaces. One interface involves the transmembrane domain (TM) 1, TM2, the C-terminal H8 and extracellular loop 1. The other interface engages residues from TM4, TM5, intracellular loop 2 and extracellular loop 2. Structural comparisons show that this ligand-free state is in an inactive conformation. This provides the structural basis of GPCR dimerization and oligomerization.

Migrastatin analogues target fascin to block tumour metastasis

Tumour metastasis is the primary cause of death of cancer patients. Development of new therapeutics preventing tumour metastasis is urgently needed. Migrastatin is a natural product secreted by Streptomyces, and synthesized migrastatin analogues such as macroketone are potent inhibitors of metastatic tumour cell migration, invasion and metastasis. Here we show that these migrastatin analogues target the actin-bundling protein fascin to inhibit its activity. X-ray crystal structural studies reveal that migrastatin analogues bind to one of the actin-binding sites on fascin. Our data demonstrate that actin cytoskeletal proteins such as fascin can be explored as new molecular targets for cancer treatment, in a similar manner to the microtubule protein tubulin.

Dosage-dependent switch from G protein-coupled to G protein-independent signaling by a GPCR

G‐protein‐coupled receptors (GPCRs) mostly signal through heterotrimeric G proteins. Increasing evidence suggests that GPCRs could function in a G‐protein‐independent manner. Here, we show that at low concentrations of an agonist, β2‐adrenergic receptors (β2‐ARs) signal through Gαs to activate the mitogen‐activated protein kinase pathway in mouse embryonic fibroblast cells. At high agonist concentrations, signals are also transduced through β2‐ARs via an additional pathway that is G‐protein‐independent but tyrosine kinase Src‐dependent. This new dosage‐dependent switch of signaling modes of GPCRs has significant implications for GPCR intrinsic properties and desensitization.