Regulation of the kinase activity of the MIK GCK-like MAP4K by alternative splicing

Alternative splicing of introns is essential to ensure the complexity of mammalian genome functions. In particular, the generation of a high number of different isoforms by alternative splicing is an important characteristic of genes coding for signalling proteins such as mitogen activated protein kinases (MAPKs). This is thought to allow these proteins to transduce multiple stimuli in a highly regulated manner. Plant genes are also subjected to alternative splicing. Nevertheless, clear examples of the functional consequences of this phenomenon are still scarce in plants. MIK is a maize gene coding for a GCK-like MAP4K that can be activated by interaction with maize atypical receptor kinase (MARK), an atypical receptor kinase. Here we show that MIK is subjected to alternative splicing. Expression of MIK leads to, at least, 4 different mature mRNAs that accumulate with particular expression profiles during maize development. Our results show that the polypeptides encoded by the different MIK mRNAs display different kinase activity and are differentially activated by interaction with the MARK receptor. Two MIK isoforms display constitutive kinase activity, one isoform is inactive but can be activated by MARK, and the fourth MIK isoform is inactive and cannot be activated by MARK. Our results constitute a clear example of the biochemical consequences of alternative splicing in plants. The selective conservation during evolution of the intron–exon structure of the region coding for the regulator domain of MIK, as well as the maintenance in maize, rice and Arabidopsis of the alternative splicing of some of these introns, are strong indications of its functional importance.     

Expression cloning of the TGF-beta type II receptor, a functional transmembrane serine/threonine kinase.

A cDNA encoding the TGF-beta type II receptor protein has been isolated by an expression cloning strategy. The cloned cDNA, when transfected into COS cells, leads to overexpression of an approximately 80 kd protein that specifically binds radioiodinated TGF-beta 1. Excess TGF-beta 1 competes for binding of radioiodinated TGF-beta 1 in a dose-dependent manner and is more effective than TGF-beta 2. The predicted receptor structure includes a cysteine-rich extracellular domain, a single hydrophobic transmembrane domain, and a predicted cytoplasmic serine/threonine kinase domain. A chimeric protein containing the intracellular domain of the type II receptor and expressed in E. coli can phosphorylate itself on serine and threonine residues in vitro, indicating that the cytoplasmic domain of the type II receptor is a functional kinase. This result implicates serine/threonine phosphorylation as an important mechanism of TGF-beta receptor-mediated signaling.     

The LIM-only protein PINCH directly interacts with integrin-linked kinase and is recruited to integrin-rich sites in spreading cells

PINCH is a widely expressed and evolutionarily conserved protein comprising primarily five LIM domains, which are cysteine-rich consensus sequences implicated in mediating protein-protein interactions. We report here that PINCH is a binding protein for integrin-linked kinase (ILK), an intracellular serine/threonine protein kinase that plays important roles in the cell adhesion, growth factor, and Wnt signaling pathways. The interaction between ILK and PINCH has been consistently observed under a variety of experimental conditions. They have interacted in yeast two-hybrid assays, in solution, and in solid-phase-based binding assays. Furthermore, ILK, but not vinculin or focal adhesion kinase, has been coisolated with PINCH from mammalian cells by immunoaffinity chromatography, indicating that PINCH and ILK associate with each other in vivo. The PINCH-ILK interaction is mediated by the N-terminal-most LIM domain (LIM1, residues 1 to 70) of PINCH and multiple ankyrin (ANK) repeats located within the N-terminal domain (residues 1 to 163) of ILK. Additionally, biochemical studies indicate that ILK, through the interaction with PINCH, is capable of forming a ternary complex with Nck-2, an SH2/SH3-containing adapter protein implicated in growth factor receptor kinase and small GTPase signaling pathways. Finally, we have found that PINCH is concentrated in peripheral ruffles of cells spreading on fibronectin and have detected clusters of PINCH that are colocalized with the alpha5beta1 integrins. These results demonstrate a specific protein recognition mechanism utilizing a specific LIM domain and multiple ANK repeats and suggest that PINCH functions as an adapter protein connecting ILK and the integrins with components of growth factor receptor kinase and small GTPase signaling pathways.     

Regulated nucleocytoplasmic transport of protein kinase D in response to G protein-coupled receptor activation

Protein kinase D (PKD)/protein kinase C mu is a serine/threonine protein kinase activated by growth factors, antigen-receptor engagement, and G protein-coupled receptor (GPCR) agonists via a phosphorylation-dependent mechanism that requires protein kinase C (PKC) activity. In order to investigate the dynamic mechanisms associated with GPCR signaling, the intracellular distribution of PKD was analyzed in live cells by imaging fluorescent protein-tagged PKD and in fixed cells by immunocytochemistry. We found that PKD shuttled between the cytoplasm and the nucleus in both fibroblasts and epithelial cells. Cell stimulation with mitogenic GPCR agonists that activate PKD induced a transient nuclear accumulation of PKD that was prevented by inhibiting PKC activity. The nuclear import of PKD requires its cys2 domain in conjunction with a nuclear import receptor, while its nuclear export requires its pleckstrin homology domain and a competent Crm1-dependent nuclear export pathway. This study thus characterizes the regulated nuclear transport of a signaling molecule in response to mitogenic GPCR agonists and positions PKD as a serine kinase whose kinase activity and intracellular localization is coordinated by PKC.     

Structure/function analysis of alpha2A-adrenergic receptor interaction with G protein-coupled receptor kinase 2

G protein-coupled receptors (GPCRs) mediate the ability of a diverse array of extracellular stimuli to control intracellular signaling. Many GPCRs are phosphorylated by G protein-coupled receptor kinases (GRKs), a process that mediates agonist-specific desensitization in many cells. Although GRK binding to activated GPCRs results in kinase activation and receptor phosphorylation, relatively little is known about the mechanism of GRK/GPCR interaction or how this interaction results in kinase activation. Here, we used the alpha2A-adrenergic receptor (alpha(2A)AR) as a model to study GRK/receptor interaction because GRK2 phosphorylation of four adjacent serines within the large third intracellular loop of this receptor is known to mediate desensitization. Various domains of the alpha(2A)AR were expressed as glutathione S-transferase fusion proteins and tested for the ability to bind purified GRK2. The second and third intracellular loops of the alpha(2A)AR directly interacted with GRK2, whereas the first intracellular loop and C-terminal domain did not. Truncation mutagenesis identified three discrete regions within the third loop that contributed to GRK2 binding, the membrane proximal N- and C-terminal regions as well as a central region adjacent to the phosphorylation sites. Site-directed mutagenesis revealed a critical role for specific basic residues within these regions in mediating GRK2 interaction with the alpha(2A)AR. Mutation of these residues within the holo-alpha(2A)AR diminished GRK2-promoted phosphorylation of the receptor as well as the ability of the kinase to be activated by receptor binding. These studies provide new insight into the mechanism of interaction and activation of GRK2 by GPCRs and suggest that GRK2 binding is critical not only for receptor phosphorylation but also for full activity of the kinase.     

Fiz1, a novel zinc finger protein interacting with the receptor tyrosine kinase Flt3.

The receptor tyrosine kinase Flt3 has been shown to play a role in proliferation and survival of hematopoietic progenitor cells as well as differentiation of early B lymphoid progenitors. However, the signaling events that control growth or differentiation are not completely understood. In order to identify new signaling molecules interacting with the cytoplasmic domain of Flt3, we performed a yeast two-hybrid screen. In addition to several SH2 domain-containing proteins, we have isolated a novel Flt3 interacting zinc finger protein (Fiz1) with 11 C(2)H(2)-type zinc fingers. Fiz1 binds to the catalytic domain of Flt3 but not to the structurally related receptor tyrosine kinases Kit, Fms, and platelet-derived growth factor receptor. This association is independent of kinase activity. The interaction between Flt3 and Fiz1 detected in yeast was confirmed by in vitro and in vivo coprecipitation assays. Fiz1 mRNA is expressed in all murine cell lines and tissues tested. Anti-Fiz1 antibodies recognize a 60-kDa protein, which is localized in the nucleus as well as in the cytoplasm. Together, these results identified a novel class of interaction between a receptor tyrosine kinase and a signaling molecule which is independent of the well established SH2 domain/phosphotyrosine binding.     

Spred1 and TESK1--two new interaction partners of the kinase MARKK/TAO1 that link the microtubule and actin cytoskeleton

The signaling from MARKK/TAO1 to the MAP/microtubule affinity-regulating kinase MARK/Par1 to phosphorylated microtubule associated proteins (MAPs) renders microtubules dynamic and plays a role in neurite outgrowth or polarity development. Because hyperphosphorylation of Tau at MARK target sites is a hallmark of Alzheimer neurodegeneration, we searched for upstream regulators by the yeast two-hybrid approach and identified two new interaction partners of MARKK, the regulatory Sprouty-related protein with EVH-1 domain1 (Spred1) and the testis-specific protein kinase (TESK1). Spred1-MARKK binding has no effect on the activity of MARKK; therefore, it does not change microtubule (MT) stability. Spred1-TESK1 binding causes inhibition of TESK1. Because TESK1 can phosphorylate cofilin and thus stabilizes F-actin stress fibers, the inhibition of TESK1 by Spred1 makes F-actin fibers dynamic. A third element in this interaction triangle is that TESK1 binds to and inhibits MARKK. Thus, in Chinese hamster ovary (CHO) cells the elevation of MARKK results in MT disruption (via activation of MARK/Par1 and phosphorylation of MAPs), but this can be blocked by TESK1. Similarly, enhanced TESK1 activity results in increased stress fibers (via phospho-cofilin), but this can be blocked by elevating Spred1. Thus, the three-way interaction between Spred1, MARKK, and TESK1 represents a pathway that links regulation of both the microtubule- and F-actin cytoskeleton.     

Identification of a switch in neurotrophin signaling by selective tyrosine phosphorylation

Neurotrophins, such as nerve growth factor and brain-derived neurotrophic factor, activate Trk receptor tyrosine kinases through receptor dimerization at the cell surface followed by autophosphorylation and recruitment of intracellular signaling molecules. The intracellular pathways used by neurotrophins share many common protein substrates that are used by other receptor tyrosine kinases (RTK), such as Shc, Grb2, FRS2, and phospholipase C-gamma. Here we describe a novel RTK mechanism that involves a 220-kilodalton membrane tetraspanning protein, ARMS/Kidins220, which is rapidly tyrosine phosphorylated in primary neurons after neurotrophin treatment. ARMS/Kidins220 undergoes multiple tyrosine phosphorylation events and also serine phosphorylation by protein kinase D. We have identified a single tyrosine (Tyr(1096)) phosphorylation event in ARMS/Kidins220 that plays a critical role in neurotrophin signaling. A reassembled complex of ARMS/Kidins220 and CrkL, an upstream component of the C3G-Rap1-MAP kinase cascade, is SH3-dependent. However, Tyr(1096) phosphorylation enables ARMS/Kidins220 to recruit CrkL through its SH2 domain, thereby freeing the CrkL SH3 domain to engage C3G for MAP kinase activation in a neurotrophin dependent manner. Accordingly, mutation of Tyr(1096) abolished CrkL interaction and sustained MAPK kinase activity, a response that is not normally observed in other RTKs. Therefore, Trk receptor signaling involves an inducible switch mechanism through an unconventional substrate that distinguishes neurotrophin action from other growth factor receptors.     

PAK5 kinase is an inhibitor of MARK/Par-1, which leads to stable microtubules and dynamic actin

MARK/Par-1 is a kinase involved in development of embryonic polarity. In neurons, MARK phosphorylates tau protein and causes its detachment from microtubules, the tracks of axonal transport. Because the target sites of MARK on tau occur at an early stage of Alzheimer neurodegeneration, we searched for interaction partners of MARK. Here we report that MARK2 is negatively regulated by PAK5, a neuronal member of the p21-activated kinase family. PAK5 suppresses the activity of MARK2 toward its target, tau protein. The inhibition requires the binding between the PAK5 and MARK2 catalytic domains, but does not require phosphorylation. In transfected Chinese hamster ovary (CHO) cells both kinases show a vesicular distribution with partial colocalization on endosomes containing AP-1/2. Although MARK2 transfected alone destabilizes microtubules and stabilizes actin stress fibers, PAK5 keeps microtubules stable through the down-regulation of MARK2 but destabilizes the F-actin network so that stress fibers and focal adhesions disappear and cells develop filopodia. The results point to an inverse relationship between actin- and microtubule-related signaling by the PAK5 and MARK2 pathways that affect both cytoskeletal networks.     

Interaction of the maize and Arabidopsis kinase interaction domains with a subset of receptor-like protein kinases: implications for transmembrane signaling in plants

The kinase interaction (KI) domain of kinase-associated protein phosphatase (KAPP) interacts with the phosphorylated form of an Arabidopsis thaliana receptor-like protein kinase (RLK). The KI domain may recruit KAPP into an RLK-initiated signaling complex. To examine additional roles that this domain may play in plant signal transduction, a search was conducted for other KI domain-containing proteins. One gene was isolated which encodes a KI domain, the maize homolog of KAPP. To test whether the maize KI domain associates with other maize proteins, it was used as a probe in a protein–protein interaction cloning strategy. A new maize RLK, K I domain i nteracting k inase 1 (KIK1), was identified by its interaction with the maize KI domain. The maize KI domain and the KIK1 kinase domain association required phosphorylation of the kinase. This work establishes that the KI domain phosphorylation-dependent signaling mechanism is present in both monocots and dicots. Additionally, it was determined that both the maize and Arabidopsis KI domains interact with several but not all of the active RLKs assayed. These multiple associations imply that KAPP may function in a number of RLK-initiated signaling pathways.     

p21-activated kinase 1 (PAK1) interacts with the Grb2 adapter protein to couple to growth factor signaling

A variety of intracellular signaling pathways are linked to cell surface receptor signaling through their recruitment by Src homology 2 (SH2)/SH3-containing adapter molecules. p21-activated kinase 1 (PAK1) is an effector of Rac/Cdc42 GTPases that has been implicated in the regulation of cytoskeletal dynamics, proliferation, and cell survival signaling. In this study, we describe the specific interaction of PAK1 with the Grb2 adapter protein both in vitro and in vivo. We identify the site of this interaction as the second proline-rich SH3 binding domain of PAK1. Stimulation of the epidermal growth factor receptor (EGFR) in HaCaT cells enhances the level of EGFR-associated PAK1 and Grb2, although the PAK1-Grb2 association is itself independent of this stimulation. A cell-permeant TAT-tagged peptide encompassing the second proline-rich SH3 binding domain of PAK1 simultaneously blocked Grb2 and activated EGFR association with PAK1, in vitro and in vivo, indicating that Grb2 mediates the interaction of PAK1 with the activated EGFR. Blockade of this interaction decreased the epidermal growth factor-induced extension of membrane lamellae. Thus Grb2 may serve as an important mechanism for linking downstream PAK signaling to various upstream pathways.     

Identification of a Src kinase SH3 binding site in the C-terminal domain of the human ErbB2 receptor tyrosine kinase

Overexpression of the ErbB2 receptor tyrosine kinase is associated with most aggressive tumors in breast cancer patients and is thus one of the main investigated therapeutic targets. Human ErbB2 C-terminal domain is an unstructured anchor that recruits specific adaptors for signaling cascades resulting in cell growth, differentiation and migration. Herein, we report the presence of a SH3 binding motif in the proline rich unfolded ErbB2 C-terminal region. NMR analysis of this motif supports a PPII helix conformation and the binding to Fyn-SH3 domain. The interaction of a kinase of the Src family with ErbB2 C-terminal domain could contribute to synergistic intracellular signaling and enhanced oncogenesis.     

Differential activation of the Ras/extracellular-signal-regulated protein kinase pathway is responsible for the biological consequences induced by the Axl receptor tyrosine kinase.

To understand the mechanism of Axl signaling, we have initiated studies to delineate downstream components in interleukin-3-dependent 32D cells by using a chimeric receptor containing the recombinant epidermal growth factor (EGF) receptor extracellular and transmembrane domains and the Axl kinase domain (EAK [for EGF receptor-Axl kinase]). We have previously shown that upon exogenous EGF stimulation, 32D-EAK cells are capable of proliferation in the absence of interleukin-3. With this system, we determined that EAK-induced cell survival and mitogenesis are dependent upon the Ras/extracellular-signal-regulated protein kinase (ERK) cascade. Although the phosphatidylinositol-3 kinase pathway is activated upon EAK signaling, it appears to be dispensable for the biological actions of the Axl kinase. Furthermore, we demonstrated that different threshold levels of Ras/ERK activation are needed to induce a block to apoptosis or proliferation in 32D cells. Recently, we have identified an Axl ligand, GAS6. Surprisingly, GAS6-stimulated 32D-Axl cells exhibited no blockage to apoptosis or mitogenic response which is correlated with the absence of Ras/ERK activation. Taken together, these data suggest that different extracellular domains dramatically alter the intracellular response of the Axl kinase. Furthermore, our data suggest that the GAS6-Axl interaction does not induce mitogenesis and that its exact role remains to be determined.     

Janus kinase 2 determinants for growth hormone receptor association,surface assembly,and signaling

GH signaling depends on functional interaction of the GH receptor (GHR) and the cytoplasmic tyrosine kinase, Janus kinase 2 (JAK2), which possesses a C-terminal kinase domain, a catalytically inactive pseudokinase domain just N-terminal to the kinase domain, and an N-terminal half shown by us and others to harbor elements for GHR association. Computational analyses indicate that JAKs contain in their N termini ( approximately 450 residues) divergent FERM domains. FERM domains (or subdomains within them) in JAKS may be important for associations with cytokine receptors. For some cytokine receptors, JAK interaction may be required for receptor surface expression. We previously demonstrated that a JAK2 mutant devoid of its N-terminal 239 residues (JAK2-Delta1-239) did not associate with GHR and could not mediate GH- induced signaling. In this report we employ a JAK2-deficient cell line to further define N-terminal JAK2 regions required for physical and functional association with the GHR. We also examine whether JAK2 expression affects cell surface expression of the GHR. Our results suggest that FERM motifs play an important role in the interaction of GHR and JAK2. While JAK2 expression is not required for detectable surface GHR expression, an increased JAK2 level increases the fraction of GHRs that achieves resistance to deglycosylation by endoglycosidase H, suggesting that the GHR-JAK2 association may enhance either the receptor's efficiency of maturation or its stability. Further, we report evidence for the existence of a novel GH-inducible functional interaction between JAK2 molecules that may be important in the mechanism of GH-triggered JAK2 signaling.     

MEK5, a new target of the atypical protein kinase C isoforms in mitogenic signaling

The MEK5-extracellular signal-regulated kinase (ERK5) tandem is a novel mitogen-activated protein kinase cassette critically involved in mitogenic activation by the epidermal growth factor (EGF). The atypical protein kinase C isoforms (aPKCs) have been shown to be required for cell growth and proliferation and have been reported to interact with the adapter protein p62 through a short stretch of acidic amino acids termed the aPKC interaction domain. This region is also present in MEK5, suggesting that it may be an aPKC-binding partner. Here we demonstrate that the aPKCs interact in an EGF-inducible manner with MEK5 and that this interaction is required and sufficient for the activation of MEK5 in response to EGF. Consistent with the role of the aPKCs in the MEK5-ERK5 pathway, we show that zetaPKC and lambda/iotaPKC activate the Jun promoter through the MEF2C element, a well-established target of ERK5. From all these results, we conclude that MEK5 is a critical target of the aPKCs during mitogenic signaling.     

PRO40 Is a Scaffold Protein of the Cell Wall Integrity Pathway,Linking the MAP Kinase Module to the Upstream Activator Protein Kinase C

Mitogen-activated protein kinase (MAPK) pathways are crucial signaling instruments in eukaryotes. Most ascomycetes possess three MAPK modules that are involved in key developmental processes like sexual propagation or pathogenesis. However, the regulation of these modules by adapters or scaffolds is largely unknown. Here, we studied the function of the cell wall integrity (CWI) MAPK module in the model fungus Sordaria macrospora. Using a forward genetic approach, we found that sterile mutant pro30 has a mutated mik1 gene that encodes the MAPK kinase kinase (MAPKKK) of the proposed CWI pathway. We generated single deletion mutants lacking MAPKKK MIK1, MAPK kinase (MAPKK) MEK1, or MAPK MAK1 and found them all to be sterile, cell fusion-deficient and highly impaired in vegetative growth and cell wall stress response. By searching for MEK1 interaction partners via tandem affinity purification and mass spectrometry, we identified previously characterized developmental protein PRO40 as a MEK1 interaction partner. Although fungal PRO40 homologs have been implicated in diverse developmental processes, their molecular function is currently unknown. Extensive affinity purification, mass spectrometry, and yeast two-hybrid experiments showed that PRO40 is able to bind MIK1, MEK1, and the upstream activator protein kinase C (PKC1). We further found that the PRO40 N-terminal disordered region and the central region encompassing a WW interaction domain are sufficient to govern interaction with MEK1. Most importantly, time- and stress-dependent phosphorylation studies showed that PRO40 is required for MAK1 activity. The sum of our results implies that PRO40 is a scaffold protein for the CWI pathway, linking the MAPK module to the upstream activator PKC1. Our data provide important insights into the mechanistic role of a protein that has been implicated in sexual and asexual development, cell fusion, symbiosis, and pathogenicity in different fungal systems.