The ROCO kinase QkgA is necessary for proliferation inhibition by autocrine signals in Dictyostelium discoideum

Abstract

AprA and CfaD are secreted proteins that function as autocrine signals to inhibit cell proliferation in Dictyostelium discoideum. Cells lacking AprA or CfaD proliferate rapidly, and adding AprA or CfaD to cells slows proliferation. Cells lacking the ROCO kinase QkgA proliferate rapidly, with a doubling time 83% of that of the wild type, and overexpression of a QkgA-green fluorescent protein (GFP) fusion protein slows cell proliferation. We found that qkgA(-) cells accumulate normal levels of extracellular AprA and CfaD. Exogenous AprA or CfaD does not slow the proliferation of cells lacking qkgA, and expression of QkgA-GFP in qkgA(-) cells rescues this insensitivity. Like cells lacking AprA or CfaD, cells lacking QkgA tend to be multinucleate, accumulate nuclei rapidly, and show a mass and protein accumulation per nucleus like those of the wild type, suggesting that QkgA negatively regulates proliferation but not growth. Despite their rapid proliferation, cells lacking AprA, CfaD, or QkgA expand as a colony on bacteria less rapidly than the wild type. Unlike AprA and CfaD, QkgA does not affect spore viability following multicellular development. Together, these results indicate that QkgA is necessary for proliferation inhibition by AprA and CfaD, that QkgA mediates some but not all of the effects of AprA and CfaD, and that QkgA may function downstream of these proteins in a signal transduction pathway regulating proliferation.

Fig. 1.

QkgA slows proliferation and lowers the stationary density of cells. (A) Log-phase cells were inoculated into HL5 medium at 1 × 10⁵ cells/ml, and cell densities were measured daily. Values are means ± SEMs (n ≥ 3) for all conditions. WT, wild type. (B) One thousand cells were plated on SM/5 plates with K. aerogenes bacteria, and the total number of cells was determined daily. By 72 h, cells had begun to overgrow the bacteria. Values are mean ± SEMs (n = 4 for all conditions). (C) Cell numbers per plate at 24 h. The absence of error bars indicates that the error was smaller than the plot symbol. *, P < 0.05; **, P < 0.01 (repeated-measures one-way ANOVA, Tukey's test). ns, not significant.

Fig. 2.

Expression of QkgA-GFP in wild-type and qkgA⁻ cells. (A) Cells of the indicated genotype were grown in low-fluorescence axenic medium for 24 h and then washed once in medium and imaged by fluorescence microscopy using a 40× objective. Scale bar, 50 μm. DIC, differential inference contrast. (B) qkgA⁻/act15::qgkA-GFP cells grown in HL5 medium were fixed in 4% paraformaldehyde and imaged with a 60× objective. The image was subsequently three-dimensionally deconvoluted. Scale bar, 1 μm.

Fig. 3.

qkgA⁻ cells secrete AprA and CfaD. Conditioned medium from wild-type and qkgA⁻ cells at log phase (3 × 10⁶ cells/ml) (A) or after log phase (12 × 10⁶ cells/ml) (B) were assayed by Western blotting with anti-AprA antibodies (left) or anti-CfaD antibodies (right). Data are representative of three independent experiments. The asterisk indicates a breakdown product of CfaD. Numbers at the left indicate molecular masses in kDa.

Fig. 4.

The expression of QkgA-GFP rescues the insensitivity of qkgA⁻ cells to rAprA (left) and rCfaD (right). Proliferating cells were incubated for 16 h with either 1,000 ng/ml rAprA, 600 ng/ml rCfaD, or an equivalent volume of buffer, and cell densities were then determined. The percent inhibition of cell density compared to that of a buffer control is shown. Values are means ± SEMs (n ≥ 4 for all conditions). *, P < 0.05; **, P < 0.01; ***, P < 0.001 (one-way ANOVA, Tukey's test).

Fig. 5.

AprA, CfaD, and QkgA affect the expansion of colonies on bacterial lawns. Serial dilutions of log-phase cells in shaking culture (1 × 10⁶ to 4 × 10⁶ cells/ml) were mixed with bacteria and spread on SM/5 plates, and the average diameters of well-spaced colonies were determined daily. Values are means ± SEMs (n ≥ 3). The absence of error bars indicates that the error was smaller than the plot symbol. For the wild type, plates were overgrown after day 8, and colony boundaries could not be determined after this point.

Fig. 6.

AprA, CfaD, and QkgA affect cell dispersal at the borders of colonies during growth with bacteria. Small spots of wild-type, aprA⁻, cfaD⁻, or qkgA⁻ cells were grown overnight in glass culture chambers with K. aerogenes bacteria, and interfaces between bacteria and Dictyostelium cell populations were imaged. Scale bar, 100 μm.