The Myotonic Dystrophy Kinase-related Cdc42-binding Kinase Is Involved in the Regulation of Neurite Outgrowth in PC12 Cells*

, The myotonic dystrophy kinase-related Cdc42-bind-ing kinase (MRCK a ) has been implicated in the morphological activities of Cdc42 in nonneural cells. Both MRCK a and the kinase-related Rho-binding kinase (ROK a ) are involved in nonmuscle myosin light-chain phosphorylation and associated actin cytoskeleton reor-ganization. We now show that in PC12 cells, overexpression of the kinase domain of MRCK a and ROK a resulted in retraction of neurites formed on nerve growth factor (NGF) treatment, as observed with RhoA. However, in-troduction of kinase-dead MRCK a did not result in NGF-independent neurite outgrowth as observed with dominant negative kinase-dead ROK a or the Rho inhibitor C3. Neurite outgrowth induced by NGF or kinase-dead ROK a was inhibited by dominant negative Cdc42 N17 , Rac1 N17 , and the Src homology 3 domain of c-Crk, indicating the participation of common downstream components. Neurite outgrowth induced by either agent was blocked by kinase-dead MRCK a lacking the p21-binding domain or by a minimal C-terminal regulatory region consisting of the cysteine-rich domain/pleckstrin homology domain plus a region with homology to citron. The latter region alone was an effective blocker of NGF-induced outgrowth. These results suggest that although ROK a is involved The Myotonic Dystrophy Kinase-related Cdc42-binding Kinase Is Involved in the

PC12 cells have been used as a model system for investigating neuronal differentiation in vitro (1,2). Their differentiation has been shown to be stimulated by nerve growth factor (NGF) 1 involving the NGF receptor trk, Ras, Raf1, and mitogen-activated protein kinase cascade (2). Recent studies on PC12 cells and other neuronal cell lines have indicated that the Rho subfamily of GTPases are key mediators of cytoskeletal changes necessary for neurite outgrowth and retraction. Cdc42 and Rac1 mediate outgrowth, whereas RhoA is responsible for retraction (3)(4)(5). Genetic studies on the Drosophila central nervous system have also implicated significant roles for Cdc42 and Rac1 in dendritic and axonal growth, respectively (6).
We have recently identified myotonin kinase-related serine/ threonine kinases MRCK␣ and ␤ (7), which interact with the GTP-bound form of Cdc42 and to a lesser extent that of Rac, which is related to the RhoA-binding kinases variously called ROK, ROCK, or Rho kinase (8 -10). The common catalytic domain of all these related kinases phosphorylates nonmuscle myosin light chain-2 at serine-19 (7,11), which is believed to play an important role in myosin contractile activity and associated changes in the organization of actin microfilaments in intact cells (12). In adherent cells in culture, ROK␣ acts downstream of RhoA in inducing stress fiber and focal adhesion formation (13)(14)(15), whereas MRCK␣ plays a role in mediating filopodia formation as a Cdc42 effector (7). GEK, the Drosophila homolog of MRCK, has also been suggested to regulate actin polymerization as a Cdc42 effector (16). In this study, we have microinjected constructs of MRCK␣ and ROK␣ and their various mutant forms into PC12 cells and investigated their effects on neurite morphology. We show that although ROK␣ can cause neurite retraction, MRCK␣ may conversely participate in neurite outgrowth downstream of Cdc42 and Rac.
Cell Culture, Microinjection, Transfection, and Cell Staining of PC12 Cells-PC 12 cells were cultured in Dulbecco's modified Eagle's medium containing 5% fetal bovine serum (Life Technologies, Inc.) and 10% horse serum (Sigma). For microinjection of nondifferentiated PC12 cells, subconfluent cells plated on collagen-coated coverslips for 48 h were microinjected with different constructs (50 ng/l). 2-4 h after injection, cells were fixed with 4% paraformaldehyde and stained with the combination of various primary antibodies: anti-HA (12CA5; Roche Molecular Biochemicals) or anti-FLAG, (M2; IBI). Stained cells were analyzed with an MRC 600 confocal imager adapted to a ZEISS Axioplan microscope. For differentiation, 50 ng/ml NGF (human recombi-* This work was supported in part by the Glaxo-Singapore Research Fund. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM /EBI Data Bank with accession number(s) AA197266.
¶ To whom correspondence should be addressed: Glaxo-IMCB Group, Institute of Molecular and Cell Biology, 30 Medical Dr., Singapore, 117609 Singapore. Tel.: 65-874-6167; Fax: 65-774-0742. 1 The abbreviations used are: MRCK, myotonic dystrophy kinaserelated Cdc42-binding kinase; ROK, RhoA-binding kinase; NGF, nerve growth factor; CRD, cysteine-rich domain; PH, pleckstrin homology; SH3, Src homology 3; HA, hemagglutinin. nant; Sigma) in serum-free medium was used. After 3 days of NGF treatment, cells were microinjected with different constructs and stained as described previously (13). For PC12 cell transfection, subconfluent cells plated on 20 g/ml laminin-coated coverslips were transfected with various HA-or FLAG-tagged DNA constructs (1 g/ml) with LipofectAMINE (Life Technologies, Inc.) according to recommended protocol. 5 h after transfection, cells were exposed to 50 ng/ml NGF in medium containing 2% serum for 48 h before fixing and staining as described above. For evaluation of neurite outgrowth, the total number of cells expressing the HA-or FLAG-tag were counted, and cells with neurite of more than two body lengths were regarded as exhibiting neurite growth. Control cells were either injected or transfected with a pXJ40-GST construct.

Neurite Retraction in NGF-treated PC12 Cells Is Induced by Overexpression of the Catalytic Domain of Either ROK␣ or
In nonneuronal cells, expression of either ROK␣ or MRCK␣, or their kinase domains, resulted in increases in stress fibers and focal adhesions (13)(14)(15). Here using PC12 cells treated with NGF for 48 h, we find that microinjection of the MRCK␣ catalytic domain construct resulted in neurite retraction (Fig.  1B) as did that of the ROK␣. Similar results were obtained with full-length ROK␣ or MRCK␣ (data not shown), which are catalytically active in vitro, phosphorylating similar substrates (7,13).
Kinase-inactive MRCK␣-KD Unlike ROK␣-KD Does not Induce Neurite Outgrowth-We have previously shown in HeLa cells and fibroblasts that the kinase-dead ROK␣-KD caused rapid losses of stress fibers and focal adhesion, which were not observed with the kinase-dead MRCK␣-KD, suggesting distinctive differences in their activity on the actin cytoskeleton (7,13). In PC12 cells, microinjection of the dominant negative ROK␣-KD construct can induce outgrowth in the absence of NGF. This effect was very similar to that observed with C3 toxin injection, which inhibits RhoA by ADP-ribosylation. However, no such effect was observed when MRCK␣-KD was expressed in these cells (Fig. 1C). We conclude that inhibition of the catalytic activity of ROK␣, but not of MRCK␣, can mimic the effects of inhibiting RhoA in PC12 cells in inducing neurite outgrowth. Unlike NGF treatment which gave obvious effects only after 1-2 days, microinjection of either ROK␣-KD or C3 toxin produced neurites within hours. These neurites were also more extensively branched than those obtained after NGF treatment (see Fig. 2A and Fig. 3B).
Neurite Outgrowth Induced by NGF and ROK␣-KD Employ Common Components-We next investigated whether the action of NGF and of ROK␣-KD in inducing neurite outgrowth involved components in common. GTPases such as Cdc42 and Rac1 (3) and the adaptor protein Crk (18,19) have been shown to be required for neurite outgrowth with their dominant negative mutants being potent inhibitors of outgrowth. As shown in Fig. 2, A and B, transfection of constructs encoding Cdc42 N17 , Rac1 N17 , and Crk-SH3 but not ACK-SH3 could effectively block NGF-induced neurite outgrowth in PC12 cells. These constructs exerted similar blocking effects on the neurite outgrowth induced by ROK␣-KD (Fig. 2, A and B). These data suggest that neurite outgrowth induced either by NGF treatment or inhibition of the RhoA pathway by ROK␣-KD expression involve common downstream components, including Cdc42 and Rac.

Mutants of MRCK␣ Including a p21-binding and Kinasedeficient Mutant and Truncated C-terminal Fragments Block both NGF-and ROK␣-KD-induced Neurite Outgrowth-As in other cells, endogenous MRCK␣ does not appear to be involved in mediating Rho-type actions in PC12 cells. In nonneural cells,
there is evidence that MRCK␣ can potentiate the effects of Cdc42 in promoting peripheral changes (7). To determine whether MRCK␣ exerted a similar peripheral effect in PC12 cells, they were transfected with various plasmid constructs of MRCK␣ (Fig. 3A) including MRCK␣-TM (which was kinase and p21-binding defective), MRCK␣-CPC (containing the cysteine-rich/PH domains as well as a region with marked homology to citron; Refs. 20 and 21), and MRCK␣-CC (containing the cysteine-rich domain/citron homology region with the PH domain deleted). The response of these transfected cells to NGF treatment was examined. In separate experiments, these constructs were also microinjected together with ROK␣-KD to assess their effects on ROK␣-KD-induced neurite outgrowth.
A substantial attenuation of NGF-induced neurite outgrowth was observed with all three mutants, MRCK␣-TM, -CPC, and -CC (Fig. 3, B and C). Strikingly, a similar effect was obtained when cells were transfected with the mutant MRCK␣-C, which contained only the C-terminal region with homology to citron (bottom photograph). MRCK␣-TM and MRCK␣-CPC also blocked neurite outgrowth induced by ROK␣-KD expression but to a lower extent, whereas MRCK␣-CC and MRCK␣-C were ineffective in this respect. These results indicate that endogenous MRCK␣ is involved in promoting neurite outgrowth induced by either NGF or ROK␣-KD. Further, they revealed the presence of functional domains in MRCK␣ including cysteinerich/PH domains and a regulatory citron homology domain. The inhibitory effects were more prominent with NGF treatment, in which the minimal citron homology domain alone was highly effective. With regard to outgrowth induced by ROK␣-KD, the PH domain of MRCK␣ appears to be crucial. The alignment of the important citron homology domain with homologous sequences in the data base is shown in Fig. 4A. Sequence similarities extended from the newly reported mammalian citron kinase (20,21), the nck-interacting kinase NIK to the yeast guanine nucleotide exchange factor ROM-2. Interestingly cysteine-rich and PH domains are often found in front of the citron homology domain at the C-terminal end of the proteins. DISCUSSION Neuronal cells in culture respond differently to various growth factors that regulate their neurite morphology (3)(4)(5). Treatment of neuroblastoma cells with lysophosphatidic acid causes neurite retraction through RhoA activation (4, 5). On the other hand, treatment with acetylcholine acting as a growth factor results in neurite growth cone filopodial and laemellipodial formation, with these morphological effects being regulated by Cdc42 and Rac, respectively (3). In PC12 cells, treatment with NGF causes neurite outgrowth (22) with Ras thought to be involved, because dominant negative Ras blocks this NGF effect (23). Recent evidence suggests that Cdc42 and Rac1 are also major players in these morphological events as their dominant negatives are potent inhibitors of neurite formation (3). In NGF-differentiated PC12 cells ROK␣ promotes retraction of neurites. This retraction is also observed when these cells are microinjected with the ROK␣ catalytic domain construct. While this work was in progress, similar findings were reported in PC12 (24) and NIE-115 (25) cells. The MRCK␣ catalytic domain elicits similar results as the ROK␣ catalytic domain clearly indicating that the overexpression of the catalytic domain of these related kinases in PC12 cells is sufficient for the retraction activity. This is not unexpected because these two kinases can act on similar substrates, with nonmuscle myosin light chain phosphorylation being most important for actomyosin contractility (7,11,12).
However, whereas expression of the kinase-dead ROK␣-KD can induce neurite outgrowth MRCK␣-KD is totally ineffective. The multipolar outgrowth pattern induced by ROK␣-KD is similar to that reported for C3 toxin (4). These results are in keeping with RhoA being an important regulator of neurite morphology (26). Activation of RhoA results in neurite retraction, whereas blocking of RhoA by C3 or kinase-dead ROK␣ promotes neurite outgrowth.
Neurite outgrowth induced by ROK␣-KD expression or by NGF treatment appears to involve common components. The outgrowth effects of both agents are blocked by the inhibitory N-terminal truncated c-Crk-II 102-304 , Cdc42 N17 and Rac1 N17 FIG. 4. A, alignment of a novel citron homology domain from rat MRCK␣, a Caenorhabditis elegans MRCK homolog (ceMRCK), mouse citron kinase (21), two other C. elegans citron-like proteins (ceLF59A6 and ceW0288), mouse NIK, and the yeast guanine exchange factor (GEF) ROM-2 from the GenBank TM data base using the DNAStar Clustal method. Identical amino acid residues are shown in black, and highly conserved residues are marked with asterisks. A diagrammatic representation of the various proteins containing the citron homology is also shown for comparison. B, a model for the regulation of neurite outgrowth in PC12 cells. Activation of RhoA in PC12 cells by serum factors such as lysophosphatidic acid (LPA) subsequently activates ROK␣ and results in neurite retraction. NGF activates Ras and Rap, which may lead to activation of Cdc42 and Rac1. Cdc42 then activates MRCK␣, whose enzymatic activity is required for neurite outgrowth. Inhibition of RhoA or of ROK␣ can also lead to neurite outgrowth requiring MRCK␣ action; it is possible that the availability/interactions of Crk may play a role in determining whether outgrowth (Ras/Rap) or retraction (RhoA) of neurites ensues. (Fig. 2, B and C), and Rap N17 (data not shown). As the c-Crk mutant was a potent inhibitor in both cases, it is possible that c-Crk (c-Crk-l in PC12 cells) provides a link between the RhoA and the NGF signaling pathways. v-Crk has been recently reported to regulate RhoA activity (27), whereas c-Crk is known to mediate Rap1 effects through the exchange factor C3G believed to be essential for sustaining Ras signaling and PC12 differentiation (28). Furthermore, a direct activation of Rac1 through c-CrkII and DOCK180 has also been documented (29,30). Blocking of the action of RhoA by C3 or ROK␣-KD could allow c-Crk to be available for alternate pathways for which Cdc42 and Rac1 are required (see model in Fig. 4B). RasGAP120 and RhoGAP190 interactions may also be crucial to the cross-talk between these two pathways (31). Whereas NGF-induced neurite outgrowth could be blocked with either PD-98059 or SB-203580 (32), which are specific inhibitors for extra cellular signal-regulated kinases and p38 mitogen-activated protein kinase, respectively, these agents were ineffective in preventing ROK␣-KD-induced neurite outgrowth (results not shown). Mitogen-activated protein kinase pathways are therefore not involved in the production of neurites through Rho inhibition, and it is possible that the different types of neurite morphologies in the PC12 cells we observe reflects the involvement of different final pathways. As RhoA inhibition is a crucial step in this process, a major step in growth factor signaling may be in fine tuning of the RhoA pathway.
The neurite outgrowth induced by either ROK␣-KD or NGF was effectively blocked by the kinase-dead and p21-binding deficient MRCK␣-TM mutant, strongly suggesting that MRCK␣ is an important component involved in the outgrowth process. Another most striking finding was that a fragment containing just the citron homology region of MRCK␣ was sufficient for blocking NGF-induced neurite outgrowth. This minimal fragment did not affect ROK␣-induced outgrowth, whose blocking requires inclusion of the PH domain in the mutants (Fig. 3). The use of these MRCK␣ mutants thus also reveals further differences between the NGF-and ROK␣-KDinduced processes. The meshwork of neurites that emanate immediately on inhibition of Rho activities suggests that the latter is important in eliciting neurite outgrowth and that the cell is already primed to respond rapidly but perhaps in a disorganized fashion. It is possible that for proper neuritic outgrowth initiated by growth factors such as NGF, apart from stimulating Cdc42 and Rac activities, the regulated dampening of Rho activities is required and that the integration of such activities may explain the relatively longer period required for cells to respond to NGF.
We conclude that in PC12 cells, the two myotonic dystrophy kinase-related kinases, MRCK␣ and ROK␣, are involved in regulating neurite morphology but with contrasting roles as depicted in Fig. 4B. ROK␣ acts downstream of RhoA in inducing neurite retraction and MRCK␣ acts downstream of Cdc42/ Rac1 in promoting neurite outgrowth. Their related catalytic domains allow MRCK␣ and ROK␣ to act on similar components of the actomyosin contractile apparatus, whereas their different p21-binding and regulatory domains are probably respon-sible for specifying their spatial and temporal effects in cells responding to growth factor stimulation. Although the regulatory domains of both kinases include CRD and PH domains, an important difference is the presence of the citron homology region in MRCK␣, but absent in ROK␣, which appears to be crucially implicated in NGF-induced neurite outgrowth. The citron homology region is also present in a number of signaling molecules in various organisms (Fig. 4A). The role of this region in macromolecular interactions and especially in defining the actions of MRCK␣ merits further investigation.