Identification of the region in Cdc42 that confers the binding specificity to activated Cdc42-associated kinase

The Rho family small G-protein Cdc42 has been implicated in a diversity of biological functions. Multiple downstream effectors have been identified. How Cdc42 discriminates the interaction with its multiple downstream effectors is not known. Activated Cdc42-associated tyrosine kinase (ACK) is a very specific effector of Cdc42. To delineate the Cdc42 signaling pathway mediated by ACK, we set about to identify the specific ACK-binding region in Cdc42. We utilized TC10, another member of the Rho family of G-proteins that is 66.7% identical to Cdc42, to construct TC10/Cdc42 chimeras for screening the specific ACK-binding region in Cdc42. A region between switch I and switch II has been identified as the specific ACK-binding (AB) region. The replacement of the AB region with the corresponding region in TC10 resulted in the complete loss of ACK-binding ability but did not affect the binding to WASP, suggesting that the AB region confers the binding specificity to ACK. On the other hand, replacement of the corresponding region of TC10 with the AB region enabled TC10 to acquire ACK-binding ability. Eight residues are different between the AB region and the corresponding region of TC10. The mutational analysis indicated that all eight residues contribute to the binding to ACK2. The assays for the Cdc42-mediated activation of ACK2 indicated that the AB region is essential for Cdc42 to activate ACK2 in cells. Thus, our studies have defined a specific ACK-binding region in Cdc42 and have provided a molecular basis for generating ACK binding-defective mutants of Cdc42 to delineate ACK-mediated signaling pathway.     

Requirement of activated Cdc42-associated kinase for survival of v-Ras-transformed mammalian cells

Activated Cdc42-associated kinase (ACK) has been shown to be an important effector molecule for the small GTPase Cdc42. We have shown previously an essential role for Cdc42 in the transduction of Ras signals for the transformation of mammalian cells. In this report, we show that the ACK-1 isoform of ACK plays a critical role in transducing Ras-Cdc42 signals in the NIH 3T3 cells. Overexpression of a dominant-negative (K214R) mutant of ACK-1 inhibits Ras-induced up-regulation of c-fos and inhibits the growth of v-Ras-transformed NIH 3T3 cells. Using small interfering RNA, we knocked down the expression of ACK-1 in both v-Ha-Ras-transformed and parental NIH 3T3 cells and found that down-regulation of ACK-1 inhibited cell growth by inducing apoptosis only in v-Ha-Ras-transformed but not parental NIH 3T3 cells. In addition, we studied the effect of several tyrosine kinase inhibitors and found that PD158780 inhibits the kinase activity of ACK-1 in vitro. We also found that PD158780 inhibits the growth of v-Ha-Ras-transformed NIH 3T3 cells. Taken together, our results suggest that ACK-1 kinase plays an important role in the survival of v-Ha-Ras-transformed cells, suggesting that ACK-1 is a novel target for therapies directed at Ras-induced cancer.     

The activation mechanism of ACK1 (activated Cdc42-associated tyrosine kinase 1)

ACK [activated Cdc42 (cell division cycle 42)-associated tyrosine kinase; also called TNK2 (tyrosine kinase, non-receptor, 2)] is activated in response to multiple cellular signals, including cell adhesion, growth factor receptors and heterotrimeric G-protein-coupled receptor signalling. However, the molecular mechanism underlying activation of ACK remains largely unclear. In the present study, we demonstrated that interaction of the SH3 (Src homology 3) domain with the EBD [EGFR (epidermal growth factor receptor)-binding domain] in ACK1 forms an auto-inhibition of the kinase activity. Release of this auto-inhibition is a key step for activation of ACK1. Mutation of the SH3 domain caused activation of ACK1, independent of cell adhesion, suggesting that cell adhesion-mediated activation of ACK1 is through releasing the auto-inhibition. A region at the N-terminus of ACK1 (Leu10-Leu14) is essential for cell adhesion-mediated activation. In the activation of ACK1 by EGFR signalling, Grb2 (growth-factor-receptor-bound protein 2) mediates the interaction of ACK1 with EGFR through binding to the EBD and activates ACK1 by releasing the auto-inhibition. Furthermore, we found that mutation of Ser445 to proline caused constitutive activation of ACK1. Taken together, our studies have revealed a novel molecular mechanism underlying activation of ACK1.     

Stimulation of M3 muscarinic receptors induces phosphorylation of the Cdc42 effector activated Cdc42Hs-associated kinase-1 via a Fyn tyrosine kinase signaling pathway

The tyrosine kinase, activated Cdc42Hs-associated kinase-1 (ACK-1), is a specific effector of the Rho family GTPase Cdc42. GTP-bound Cdc42 has been shown to facilitate neurite outgrowth elicited by activation of muscarinic cholinergic receptors (mAChRs). Because tyrosine kinase activity is a requirement for neuritogenesis in several cell systems, we investigated whether endogenous mAChRs (principally of the M3 subtype) expressed in human SH-SY5Y neuroblastoma cells would signal to ACK-1. Incubation of cells with the cholinergic agonist oxotremorine-M (Oxo-M) induced an approximately 6-fold increase in the tyrosine phosphorylation of ACK-1 which was inhibited by atropine. ACK-1 phosphorylation was blocked by Clostridium difficile toxin B, an inhibitor of Rho family GTPases. In contrast, disruption of the actin cytoskeleton with cytochalasin D stimulated ACK-1 phosphorylation, and moreover, addition of Oxo-M to cells preincubated with this agent elicited a further increase in phosphorylation, indicating that an intact cytoskeleton is not required for mAChR signaling to ACK-1. Although stimulation of M3 mAChRs induces both an increase in intracellular Ca2+ and activation of protein kinase C (PKC), neither of these second messenger pathways was required for receptor-stimulated ACK-1 phosphorylation. Instead, inhibition of PKC resulted in a 2-fold increase in Oxo-M-stimulated ACK-1 phosphorylation, whereas acute activation of PKC with phorbol ester decreased ACK-1 phosphorylation. The agonist-induced tyrosine phosphorylation of ACK-1 was blocked by inhibitors of Src family kinases, and ACK-1 was coprecipitated with Fyn (but not Src) in an agonist-dependent manner. Finally, scrape loading cells with glutathione S-transferase fusion proteins of either the Fyn-SH2 or Fyn-SH3 domain significantly attenuated mAChR-stimulated ACK-1 tyrosine phosphorylation. The data are the first to show phosphorylation of ACK-1 after stimulation of a receptor coupled to neurite outgrowth and indicate that a Rho family GTPase (i.e. Cdc42) and Fyn are essential upstream elements of this signaling pathway.     

Cdc42 antagonizes inductive action of cAMP on cell shape,via effects of the myotonic dystrophy kinase-related Cdc42-binding kinase (MRCK) on myosin light chain phosphorylation

Rho GTPases play pivotal roles in regulating cell morphology. We previously showed that RhoA acts via ROKalpha to counteract the effects of the classical second messenger cyclic AMP on cell shape changes. Here we show that active Cdc42V12 also competes against the cAMP-induced stellate morphology in SH-EP cells. This Cdc42 effect is not mediated by the RhoA/ ROK pathway but rather the related MRCKalpha, a myotonic dystrophy kinase-related Cdc42-binding kinase. Co-expression of a dominant inhibitory MRCKalpha mutant with Cdc42V12 blocks the ability of the GTPase to counteract cAMP, suggesting that MRCK acts downstream of Cdc42 in this process. Cdc42V12 enhances the phosphorylation of myosin light chain (MLC) at the cell periphery and sustains focal adhesion complexes, while MLC kinase inhibitors destroy focal adhesion complexes and impair the Cdc42V12 protective effect. The data suggest that the maintenance of focal adhesion complexes via the regulation of myosin II activity underlies the ability of Cdc42 to protect against the effect of elevated cAMP.     

Activation of the Cdc42-associated tyrosine kinase-2 (ACK-2) by cell adhesion via integrin beta1

Activated Cdc42-associated kinase-2 (ACK-2) is a non-receptor tyrosine kinase that appears to be a highly specific target for the Rho-related GTP-binding protein Cdc42. In order to understand better how ACK-2 activity is regulated in cells, we have expressed epitope-tagged forms of this tyrosine kinase in COS-7 and NIH3T3 cells. We find that ACK-2 can be activated by cell adhesion in a Cdc42-dependent manner. However, unlike the focal adhesion kinase, which also is activated by cell adhesion, the activation of ACK-2 is F-actin-independent and does not require cell spreading. In addition, overexpression of ACK-2 in COS-7 cells did not result in the stimulation of extracellular signal-regulated kinase activity but rather activated the c-Jun kinase. Both anti-integrin beta1 antibody and RGD peptides inhibited the activation of ACK-2 by cell adhesion. In addition, ACK-2 was co-immunoprecipitated with integrin beta1. Overall, these findings suggest that ACK-2 interacts with integrin complexes and mediates cell adhesion signals in a Cdc42-dependent manner.     

Stimulation of Ras guanine nucleotide exchange activity of Ras-GRF1/CDC25(Mm) upon tyrosine phosphorylation by the Cdc42-regulated kinase ACK1

Ras-GRF1 is a brain-specific guanine nucleotide exchange factor (GEF) for Ras, whose activity is regulated in response to Ca(2+) influx and G protein-coupled receptor signals. In addition, Ras-GRF1 acts as a GEF for Rac when tyrosine-phosphorylated following G protein-coupled receptor stimulation. However, the mechanisms underlying the regulation of Ras-GRF1 functions remain incompletely understood. We show here that activated ACK1, a nonreceptor tyrosine kinase that belongs to the focal adhesion kinase family, causes tyrosine phosphorylation of Ras-GRF1. On the other hand, kinase-deficient ACK1 exerted no effect. GEF activity of Ras-GRF1 toward Ha-Ras, as defined by in vitro GDP binding and release assays, was augmented after tyrosine phosphorylation by ACK1. In contrast, GEF activity toward Rac1 remained latent, implying that ACK1 does not represent a tyrosine kinase that acts downstream of G protein-coupled receptors. Consistent with enhanced Ras-GEF activity, accumulation of the GTP-bound form of Ras within the cell was shown through the use of Ras-binding domain pull-down assays. Furthermore, Ras-dependent activation of ERK2 by Ras-GRF1 was enhanced following co-expression of activated ACK1. These results implicate ACK1 as an upstream modulator of Ras-GRF1 and suggest a signaling cascade consisting of Cdc42, ACK1, Ras-GRF1, and Ras in neuronal cells.     

Cdc2 and Cdk2 kinase activated by transforming growth factor-beta1 trigger apoptosis through the phosphorylation of retinoblastoma protein in FaO hepatoma cells.

The signaling pathway leading to TGF-beta1-induced apoptosis was investigated using a TGF-beta1-sensitive hepatoma cell line, FaO. Cell cycle analysis demonstrated that the accumulation of apoptotic cells was preceded by a progressive decrease of the cell population in the G(1) phase concomitant with a slight increase of the cell population in the G(2)/M phase in response to TGF-beta1. TGF-beta1 induced a transient increase in the expression of Cdc2, cyclin A, cyclin B, and cyclin D1 at an early phase of apoptosis. During TGF-beta1-induced apoptosis, the transient increase in cyclin-dependent kinase (Cdk) activities coincides with a dramatic increase in the hyperphosphorylated forms of RB. Treatment with roscovitine or olomoucine, inhibitors of Cdc2 and Cdk2, blocked TGF-beta1-induced apoptosis by inhibiting RB phosphorylation. Overexpression of Bcl-2 or adenovirus E1B 19K suppressed TGF-beta1-induced apoptosis by blocking the induction of Cdc2 mRNA and the subsequent activation of Cdc2 kinase, whereas activation of Cdk2 was not affected, suggesting that Cdc2 plays a more critical role in TGF-beta1-induced apoptosis. In conclusion, we present the evidence that Cdc2 and Cdk2 kinase activity transiently induced by TGF-beta1 phosphorylates RB as a physiological target in FaO cells and that RB hyperphosphorylation may trigger abrupt cell cycle progression, leading to irreversible cell death.     

Characterization of calcium-activated neutral protease (CANP)-associated protein kinase from bovine brain and its phosphorylation of neurofilaments

Calcium-activated neutral protease with low affinity for calcium (CANP II, Mr 76,000) can be purified to apparent homogeneity by casein affinity chromatography but contains cyclic-AMP dependent protein kinase activity. CANP II-associated kinase from bovine brain copurifies with protease activity through multiple chromatographic procedures but can be separated by cyclic-AMP affinity chromatography. Isolated protein kinase has subunits of Mr 80,000, 53,000 and 42,000. The kinase preferentially "autophosphorylates" CANP II, but histones, phosphorylase b and neurofilament proteins are also good substrates. The concentrations for half-maximal phosphorylation activity (Km) of cyclic-AMP, (32P)ATP and Mr 150,000 neurofilament protein substrate are 0.2, 6.0 and 0.5 microM, respectively. The specific activity of CANP II associated kinase in phosphorylating neurofilament proteins is intermediate between that of neurofilament- and MAPs 2-associated kinases.     

Regulation of Xenopus p21-activated kinase (X-PAK2) by Cdc42 and maturation-promoting factor controls Xenopus oocyte maturation

Signal transduction cascades involved in regulation of the cell cycle machinery are poorly understood. In the Xenopus oocyte model, meiotic maturation is triggered by MPF, a complex of p34(cdc2)-cyclin B, which is activated in response to a progesterone signal by largely unknown mechanisms. We have previously shown that the p21-activated kinase (PAK) family negatively regulates the MPF amplification loop. In this study, we identify the endogenous PAK2 as a key enzyme in this regulation and describe the pathways by which PAK2 is regulated. We show that the small GTPase Cdc42 is required for maintenance of active endogenous X-PAK2 in resting stage VI oocytes, whereas Rac1 is not involved in this regulation. During the process of maturation, X-PAK2 phosphorylation results in its inactivation and allows maturation to proceed to completion. Activation of mitogen-activated protein kinase and cyclin B-p34(cdc2) is coincident with X-PAK2 inactivation, and purified active MPF inhibits X-PAK2, demonstrating the existence of a new positive feedback loop. Our results confirm and extend the importance of p21-activated kinases in the control of the G(2)/M transition. We hypothesize that the X-PAK2/Cdc42 pathway could link p34(cdc2) activity to the major cytoskeleton rearrangements leading to spindle migration and anchorage to the animal pole cortex.     

Involvement of histone H3 (Ser10) phosphorylation in chromosome condensation without Cdc2 kinase and mitogen-activated protein kinase activation in pig oocytes

When oocytes resume meiosis, chromosomes start to condense and Cdc2 kinase becomes activated. However, recent findings show that the chromosome condensation does not always correlate with the Cdc2 kinase activity in pig oocytes. The objectives of this study were to examine 1) the correlation between chromosome condensation and histone H3 phosphorylation at serine 10 (Ser10) during the meiotic maturation of pig oocytes and 2) the effects of protein phosphatase 1/2A (PP1/ PP2A) inhibitors on the chromosome condensation and the involvement of Cdc2 kinase, MAP kinase, and histone H3 kinase in this process. The phosphorylation of histone H3 (Ser10) was first detected in the clump of condensed chromosomes at the diakinesis stage and was maintained until metaphase II. The kinase assay showed that histone H3 kinase activity was low in oocytes at the germinal vesicle stage (GV) and increased at the diakinesis stage and that high activity was maintained until metaphase II. Treatment of GV-oocytes with okadaic acid (OA) or calyculin-A (CL-A), the PP1/PP2A inhibitors, induced rapid chromosome condensation with histone H3 (Ser10) phosphorylation after 2 h. Both histone H3 kinase and MAP kinase were activated in the treated oocytes, although Cdc2 kinase was not activated. In the oocytes treated with CL-A and the MEK inhibitor U0126, neither Cdc2 kinase nor MAP kinase were activated and no oocytes underwent germinal vesicle breakdown (GVBD), although histone H3 kinase was still activated and the chromosomes condensed with histone H3 (Ser10) phosphorylation. These results suggest that the phosphorylation of histone H3 (Ser10) occurs in condensed chromosomes during maturation in pig oocytes. Furthermore, the chromosome condensation is correlated with histone H3 kinase activity but not with Cdc2 kinase and MAP kinase activities.     

Checkpoint kinase 2 (Chk2) monomers or dimers phosphorylate Cdc25C after DNA damage regardless of threonine 68 phosphorylation

We have purified and characterized human Chk2 both from baculovirus-infected insect cells and from either untreated or DNA damage-stressed human HCT116 cells. Chk2 from unstressed human cells is largely monomeric and inactive in phosphorylating its substrate, Cdc25C. It is also unphosphorylated at Thr-68, a site that is the target of the ataxia telangiectasia-mutated protein kinase. After treatment of HCT116 cells with a radiomimetic compound neocarzinostatin, active Chk2 exists as stable Thr-68-phosphorylated dimers as well as interconvertable Thr-68-unphosphorylated monomers and dimers. Interestingly, Chk2 from insect cells behaves by all criteria tested like active Chk2 from neocarzinostatin-treated HCT116 cells. Based on Stokes radius and sedimentation coefficient values, Chk2 monomers and dimers have asymmetric rather than globular shapes. Both Thr-68-phosphorylated and Thr-68-unphosphorylated forms of active Chk2 are capable of phosphorylating Cdc25C. Thus, although phosphorylation of Thr-68 may be required for initial oligomerization and activation of Chk2, it is not needed for maintenance of dimerization or kinase activity.     

Smads 2 and 3 are differentially activated by transforming growth factor-beta (TGF-beta ) in quiescent and activated hepatic stellate cells. Constitutive nuclear localization of Smads in activated cells is TGF-beta-independent

Hepatic stellate cells are the primary cell type responsible for matrix deposition in liver fibrosis, undergoing a process of transdifferentiation into fibrogenic myofibroblasts. These cells, which undergo a similar transdifferentiation process when cultured in vitro, are a major target of the profibrogenic agent transforming growth factor-beta (TGF-beta). We have studied activation of the TGF-beta downstream signaling molecules Smads 2, 3, and 4 in hepatic stellate cells (HSC) cultured in vitro for 1, 4, and 7 days, with quiescent, intermediate, and fully transdifferentiated phenotypes, respectively. Total levels of Smad4, common to multiple TGF-beta superfamily signaling pathways, do not change as HSC transdifferentiate, and the protein is found in both nucleus and cytoplasm, independent of treatment with TGF-beta or the nuclear export inhibitor leptomycin B. TGF-beta mediates activation of Smad2 primarily in early cultured cells and that of Smad3 primarily in transdifferentiated cells. The linker protein SARA, which is required for Smad2 signaling, disappears with transdifferentiation. Additionally, day 7 cells demonstrate constitutive phosphorylation and nuclear localization of Smad 2, which is not affected by pretreatment with TGF-beta-neutralizing antibodies, a type I TGF-beta receptor kinase inhibitor, or activin-neutralizing antibodies. These results demonstrate essential differences between TGF-beta-mediated signaling pathways in quiescent and in vitro transdifferentiated hepatic stellate cells.     

Epinephrine increases phosphorylation of MAP-2c in rat pheochromocytoma cells (PC12 Cells) via a protein kinase C- and mitogen activated protein kinase-dependent mechanism

Adrenoceptors mediate effects of endogenous catecholamines and have been shown to affect the neuronal development. Microtubule-associated protein-2 (MAP-2) is an important cytoskeleton protein whose phosphorylation in response to extracellular signal is involved in the regulation of neurite outgrowth and neuronal plasticity. The present study was designed to determine the effect of activation of adrenoceptor by epinephrine on MAP-2 phosphorylation in differentiation PC12 cells and, if so, to explore the mediating mechanism. We found that epinephrine could significantly increase the phosphorylation of MAP-2c at ser136 in a dose- and time-dependent manner in differentiated PC12 cells as well as microtubule arrays. Differentiated PC12 cells express alpha 2A-adrenoceptor, whose antagonists could block these mentioned effects of epinephrine, and clonidine which is the agonist of alpha 2-adrenoceptor could mimic the effect of epinephrine. Moreover phosphorylation of ERK and PKC was induced by epinephrine, and ERK and PKC specific inhibitors concentration-dependently prevented epinephrine-induced phosphorylation of MAP-2c at ser136. In addition, pretreatment of PC12 cells with epinephrine partly inhibited 30 microM nocodazole induced neurites retraction. These findings suggest that epinephrine induces phosphorylation of MAP-2c at ser136 through a alpha 2-adrenoceptor mediated, ERK/PKC-dependent signaling pathway, which may contribute to the stabilization of neurites.     

Shear stress stimulates phosphorylation of eNOS at Ser(635) by a protein kinase A-dependent mechanism

Shear stress stimulates nitric oxide (NO) production by phosphorylating endothelial NO synthase (eNOS) at Ser(1179) in a phosphoinositide-3-kinase (PI3K)- and protein kinase A (PKA)-dependent manner. The eNOS has additional potential phosphorylation sites, including Ser(116), Thr(497), and Ser(635). Here, we studied these potential phosphorylation sites in response to shear, vascular endothelial growth factor (VEGF), and 8-bromocAMP (8-BRcAMP) in bovine aortic endothelial cells (BAEC). All three stimuli induced phosphorylation of eNOS at Ser(635), which was consistently slower than that at Ser(1179). Thr(497) was rapidly dephosphorylated by 8-BRcAMP but not by shear and VEGF. None of the stimuli phosphorylated Ser(116). Whereas shear-stimulated Ser(635) phosphorylation was not affected by phosphoinositide-3-kinase inhibitors wortmannin and LY-294002, it was blocked by either treating the cells with a PKA inhibitor H89 or infecting them with a recombinant adenovirus-expressing PKA inhibitor. These results suggest that shear stress stimulates eNOS by two different mechanisms: 1) PKA- and PI3K-dependent and 2) PKA-dependent but PI3K-independent pathways. Phosphorylation of Ser(635) may play an important role in chronic regulation of eNOS in response to mechanical and humoral stimuli.     

Constitutive tyrosine phosphorylation of the T-cell receptor (TCR) zeta subunit: regulation of TCR-associated protein tyrosine kinase activity by TCR zeta.

The T-cell receptor (TCR) zeta subunit is an important component of the TCR complex, involved in signal transduction events following TCR engagement. In this study, we showed that the TCR zeta chain is constitutively tyrosine phosphorylated to similar extents in thymocytes and lymph node T cells. Approximately 35% of the tyrosine-phosphorylated TCR zeta (phospho zeta) precipitated from total cell lysates appeared to be surface associated. Furthermore, constitutive phosphorylation of TCR zeta in T cells occurred independently of antigen stimulation and did not require CD4 or CD8 coreceptor expression. In lymph node T cells that constitutively express tyrosine-phosphorylated TCR zeta, there was a direct correlation between surface TCR-associated protein tyrosine kinase (PTK) activity and expression of phospho zeta. TCR stimulation of these cells resulted in an increase in PTK activity that coprecipitated with the surface TCR complex and a corresponding increase in the levels of phospho zeta. TCR ligations also contributed to the detection of several additional phosphoproteins that coprecipitated with surface TCR complexes, including a 72-kDa tyrosine-phosphorylated protein. The presence of TCR-associated PTK activity also correlated with the binding of a 72-kDa protein, which became tyrosine phosphorylated in vitro kinase assays, to tyrosine phosphorylated TCR zeta. The cytoplasmic region of the TCR zeta chain was synthesized, tyrosine phosphorylated, and conjugated to Sepharose beads. Only tyrosine-phosphorylated, not nonphosphorylated, TCR zeta beads were capable of immunoprecipitating the 72-kDa protein from total cell lysates. This 72-kDa protein is likely the murine equivalent of human PTK ZAP-70, which has been shown to associate specifically with phospho zeta. These results suggest that TCR-associated PTK activity is regulated, at least in part, by the tyrosine phosphorylation status of TCR zeta.     

Characterization of the interaction of phorbol esters with the C1 domain of MRCK (myotonic dystrophy kinase-related Cdc42 binding kinase) alpha/beta

C1 domains mediate the recognition and subsequent signaling response to diacylglycerol and phorbol esters by protein kinase C (PKC) and by several other families of signal-transducing proteins such as the chimerins or RasGRP. MRCK (myotonic dystrophy kinase-related Cdc42 binding kinase), a member of the dystrophia myotonica protein kinase family that functions downstream of Cdc42, contains a C1 domain with substantial homology to that of the diacylglycerol/phorbol ester-responsive C1 domains and has been reported to bind phorbol ester. We have characterized here the interaction of the C1 domains of the two MRCK isoforms alpha and beta with phorbol ester. The MRCK C1 domains bind [20-(3)H]phorbol 12,13-dibutyrate with K(d) values of 10 and 17 nm, respectively, reflecting 60-90-fold weaker affinity compared with the protein kinase C delta C1b domain. In contrast to binding by the C1b domain of PKCdelta, the binding by the C1 domains of MRCK alpha and beta was fully dependent on the presence of phosphatidylserine. Comparison of ligand binding selectivity showed resemblance to that by the C1b domain of PKCalpha and marked contrast to that of the C1b domain of PKCdelta. In intact cells, as in the binding assays, the MRCK C1 domains required 50-100-fold higher concentrations of phorbol ester for induction of membrane translocation. We conclude that additional structural elements within the MRCK structure are necessary if the C1 domains of MRCK are to respond to phorbol ester at concentrations comparable with those that modulate PKC.     

Functional expression in mammalian cells of a full-length cDNA coding for the pp42/MAP kinase (p42mapk) protein.

We recently cloned from a mouse 3T3 cell cDNA library a cDNA with sequence similarity to the p42mapk protein and other members of the MAP kinase family. To determine with certainty which member of the family this clone encodes, we have expressed the cDNA in COS cells and characterized the protein product. When the pSV2MAP plasmid carrying the full-length clone was transfected into COS cells, a protein of 42,000 Da was expressed. This 42 kDa protein displayed chromatographic properties indistinguishable from the endogenous p42mapk, and could be separated from the closely related pp44. In addition, upon serum stimulation, the 42 kDa protein became tyrosine-phosphorylated and enzymatically active towards the substrate myelin basic protein. We conclude that this clone codes for a functional p42mapk protein kinase.